Hepatocyte growth factor fragments that function as potent met receptor agonists and antagonists

ABSTRACT

The NK1 fragment of hepatocyte growth factor (HGF) binds to and activates the Met receptor, a transmembrane receptor tyrosine kinase that plays a critical role in embryonic development and organ formation. The instant application discloses NK1 variant polypeptides which act as agonists or antagonists of HGF. Further disclosed are covalently linked NK1 variant polypeptides. Many of the disclosed variant polypeptides possess improved stability characteristics.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.15/365,514, filed Nov. 30, 2016, which is a continuation of U.S. patentapplication Ser. No. 13/636,053, filed Dec. 28, 2012, which is aNational Phase of International Patent Application No.PCT/US2011/029271, filed Mar. 21, 2011, which claims priority to U.S.Provisional Patent Application, Ser. No. 61/315,794, filed Mar. 19, 2010and U.S. Provisional Patent Application, Ser. No. 61/411,080, filed Nov.8, 2010, the contents of which are incorporated in their entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with Government support under contracts CA131706and CA151706 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The invention pertains to the field of polypeptide variants, inparticular variants of Hepatocyte Growth Factor.

BACKGROUND OF THE INVENTION

Hepatocyte Growth Factor (HGF), also known as Scatter Factor (SF), is amulti-functional heterodimeric protein produced predominantly bymesenchymal cells and is an effector of cells expressing the Mettyrosine kinase receptor (“c-Met”)(Bottaro et al. (1991) SCIENCE 251:802-804, Rubin et al. (1993) BIOCHIM. BIOPHYS. ACTA 1155: 357-371).Mature HGF contains two polypeptide chains, the α-chain and the β-chain.Published studies suggest it is the α-chain that contains HGF's c-Metreceptor binding domain.

Mature HGF contains two polypeptide chains, the α-chain and the β-chain.Upon binding to its cognate receptor, HGF mediates a number of cellularactivities. The HGF-Met signaling pathway plays a role in liverregeneration, wound healing, neural regeneration, angiogenesis andmalignancies. See, e.g., Cao et al. (2001) PROC. NATL. ACAD. SCI. USA98: 7443-7448, Burgess et al. (2006) CANCER RES. 66: 1721-1729, and U.S.Pat. Nos. 5,997,868 and 5,707,624.

Dysregulation of cell signaling pathways that mediate proliferation,survival, and migration are an underlying cause of many cancers. Inparticular, dysregulation and over-expression of the Met tyrosine kinasereceptor correlates to poor prognosis in many human tumors, making it anattractive target for therapeutic intervention.

There are currently no FDA approved therapeutics targeting the Metreceptor, however, a few candidate molecules are in various stages ofclinical trials. As such, molecules that potently inhibit Met receptoractivation may have a significant impact on cancer therapy. In addition,studies to develop Met-targeted molecular imaging agents fornon-invasive visualization of Met expression in vivo have been extremelylimited compared to other cancer targets. The availability of suchimaging agents would aid in cancer diagnosis, staging, and diseasemanagement, as well as help identify patients who would be goodcandidates for Met-targeted therapies.

The present invention provides potent activators and inhibitors of theMet receptor, methods for their production and use.

SUMMARY OF THE INVENTION

Hepatocyte growth factor is active in numerous tissues throughout thebody, participating in the regulation of angiogenesis, organogenesis,tissue repair and neural induction. HGF induces random movement(“scatter”) when applied to epithelial cells as well as dissociation,migration, and invasion of cells through the extracellular matrix invivo. HGF is mitogenic (induces proliferation) in many normal celltypes, including epithelial cells, vascular endothelial cells, andmelanocytes.

HGF is also a morphogen that induces transition of epithelial cells intoa mesenchymal (connective tissue-type) morphology and formation ofbranched tube-like structures; these cellular responses reflect thiscytokine's role in organogenesis and tissue repair.

HGF is cytoprotective by virtue of its anti-apoptotic activity andexerts anti-fibrotic effects by opposing TGFβ1-Smad signaling.

Each of these biological effects exerted by HGF is triggered bystimulating its cell surface receptor c-Met with concomitant activationof downstream effector pathways

The Met receptor is the product of the protooncogene met. Met is thecognate receptor for HGF. Dysregulation of HGF-Met signaling results ina phenotype of invasion and metastasis in many human tumors. Cell linesengineered to express high levels of mutated Met or wild-type Met andHGF (autocrine signaling) become tumorigenic and metastatic. Inaddition, transgenic mouse models that express Met, HGF, or mutated Met,develop different types of tumors and metastatic lesions.

Met overexpression has been found in many human cancers, includingcolorectal cancer, oral squamous cell carcinoma, hepatocellularcarcinoma, renal cell carcinoma, breast carcinoma and lung carcinoma.Since Met overexpression correlates to poor clinical prognosis, thisreceptor is an attractive target for cancer diagnosis and therapy.

The development of clinical agents that target Met has been difficult.Monoclonal antibodies cross-link the Met receptor, resulting in receptorsignalling, while kinase domain inhibitors lack target specificity.

In previous approaches, novel strategies to create hepatocyte growthfactor (HGF) or HGF fragment-based antagonists have been promising andhave highlighted the potential to develop potent therapeutics byinhibiting HGF-mediated Met activation. While these molecules opened upnew research directions for testing and generating new cancer biologics,they have not advanced to clinical trials due to limitations in Metreceptor binding affinity, recombinant expression yield, and/or proteinstability.

Alternatively, agonists of c-Met should promote tissue repair and organregeneration in two ways: first, as a prophylactic, by protectinghealthy cells from both necrotic and apoptotic death; and second, as atherapeutic, by promoting appropriate cell proliferation and migrationneeded for repair of pre-existing tissue injury. HGF agonists offer newtherapeutic approaches to protecting major organs (including the liver,kidney, lung, heart, brain, and spinal cord) from injury, and reducingfibrosis in the liver, kidney, lung, and heart.

The present invention provides a solution to these problems in theprovision of polypeptide variants of Hepatocyte Growth Factor. Invarious embodiments, variant HGF polypeptides are provided that formcovalently linked dimers. These covalent dimers result from theintroduction of novel sulfhydryl reactive groups.

In addition to the polypeptide variants, the invention provides methodsof tissue regeneration using the variants. The method includescontacting cells with a therapeutically or prophylatically effectiveamount of a HGF variant of the invention.

There is also a need for therapeutic agents that modulate the activityof Met. Accordingly, the present invention also provides HGF variants ina pharmaceutical formulation.

Other advantages, aspects and objects of the invention are apparent fromthe detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: HGF domain structure. N: N-terminal PAN module; K: Kringledomain; SPH: serine protease homology domain. Black arrow indicatescleavage site to cleave HGF into its two-chain active form. The α- andβ-chains are connected through a disulfide bond. The N-terminal andfirst Kringle domain comprise the NK1 fragment of HGF.

FIG. 2A-FIG. 2B: Yeast display construct pTMY-HA. (A) Open reading frameof pTMY-HA. Protein is displayed with a free N-terminus and linked toAga2p through its C-terminus. (B) Schematic of yeast surface display.The protein of interest (NK1) is tethered to yeast cell wall throughgenetic linkage to the N-terminus of Aga2p. Antibodies against the HAepitope tags were used to monitor cell surface expression, andinteractions with a binding partner (in this case Met-Fc) were alsomonitored.

FIG. 3: Outline of NK1 engineering strategy. In first round of directedevolution (M1) library was screened for functional binding to Met; forsecond round (M2) library was screened in parallel for either enhancedaffinity or enhance stability; for third round (M3) the M2 products wereshuffled and screened simultaneously for improved affinity andstability.

FIG. 4A-FIG. 4B: Yeast-displayed wild-type NK1 does not bind to Met. (A)Relative binding by yeast-displayed NK1 I1 to soluble Met-Fc A488 in theabsence (top) or presence (bottom) of 2 μM heparin. Levels of binding isunchanged for 20 or 200 nM Met-Fc. (B) Relative binding ofyeast-displayed NK1 I1 following heating to various temperatures.Background binding levels observed in (A) are unchanged even followingheating to 70° C.

FIG. 5A-FIG. 5B: Sort progression for round 1 (M1) and round 2 (M2) ofdirected evolution. (A) Sort progression for M1. Mutants were sorted byflow cytometry using either 20° C. or 30° C. expression temperature.Following six rounds of sorting, the bulk library population exhibiteddetectable binding to Met-Fc A488. (B) Sort progression for M2, denotingAffinity and Stability sort performed in parallel. Affinity sorts werescreened for improved binding to decreasing concentrations of Met-FcA488, while Stability sorts were screened for binding to 100 nM Met-FcA488 and improved expression at 37° C. Temperatures in parenthesisdenote the yeast surface expression temperature used for the particularsort.

FIG. 6: Sort progression for round 3 (M3) of directed evolution. Librarywas sorted in parallel for improved affinity and stability by screeningfor improved binding to decreasing concentrations of Met-Fc A488 using37° C. expression temperature. Sort 7 was conducted by staining with 2nM Met-Fc A488 following by a two day unbinding step (off) in thepresence of excess competitor.

FIG. 7A-FIG. 7B: Recombinant expression of wild-type NK1 I1, M2.1, andM2.2 in P. pastoris. (A) Western blot of supernatants followingexpression at 30° C. (B) Coomassie stained gel of purified NK1 I1, M2.1,and M2.2.

FIG. 8A-FIG. 8C: Thermal stability of NK1 mutants. (A) Yeastsurface-displayed M2.1 and M2.2. Wild-type NK1 was not functionallyexpressed on the yeast surface, so stability could not be assessed inthis manner. (B) Thermal stability of soluble NK1 proteins as determinedby variable temperature CD scans.

FIG. 9: Stability of wild-type and mutant NK1 proteins. NK1 proteinsanalyzed by size exclusion chromatography under reduced saltconcentrations (137 mM NaCl). Inset, close-up trace of wild-type NK1.

FIG. 10: Cellular activity of wild-type and mutant NK1 proteins.Agonistic activity of HGF (0.1 nM) or NK1 proteins (100 nM) was measuredin a MDCK scatter assay in the presence of 2 μM heparin. Scale bar=500μm.

FIG. 11A-FIG. 11C: Agonistic activity of wild-type and mutant NK1proteins. (A) MDCK scatter assay by HGF (0.1 nM) or NK1 proteins (100nM) in the absence of heparin. (B) Urokinase-type plasminogen activator(uPA) induced by HGF (1 nM) or NK1 proteins (100 nM) in the absence ofheparin. (C) MDCK scatter assay, testing a range of concentrations ofwild-type NK1, M2.2 D127N, or M2.2 D127K in the absence (top) orpresence (bottom) of 2 μM heparin. Images are representative ofexperiments performed on separate days. Scale bar: 500 μM.

FIG. 12: Antagonistic activity of wild-type and mutant NK1 proteins.Inhibition of HGF-induced MDCK scatter (0.1 nM HGF) by NK1 mutants (250nM) in the absence (top) or presence (bottom) of 2 μM heparin. NK1 N127Ais a previously reported antagonist based on wild-type NK1. Images arerepresentative of experiments performed on separate days. Scale bar: 500μM.

FIG. 13: Cellular activity of wild-type and mutant NK1 proteins.Inhibition of HGF-induced activity (0.1 nM) was measured in a MDCKscatter assay with NK1 proteins (250 nM) formulated with a 2:1 molarratio of heparin. Untreated MDCK cells were used as a negative control.Images are representative of experiments performed on separate days.Scale bar=500 μm.

FIG. 14A-FIG. 14B: Expression of NK1 mutants on the yeast surface andbinding to fluorescent Met-Fc as measured by flow cytometry. (A) Bindingof soluble Met-Fc conjugated to Alexa-488 (Met-Fc A488) toyeast-displayed NK1 mutants from the second (M2.1, M2.2) and third(M3S7.X.XX) rounds of directed evolution. [Met-Fc A488]: 20 nM (black),0.2 nM (grey) and 0.04 (white). M2.1 is a clone from round 2 thatcontained five of the eight most commonly occurring mutations, and M2.2was generated by site-directed mutagenesis comprising all eight of themost common mutations from the second round of directed evolution. Eachof the mutants from the third round of directed evolution binds morestrongly than M2.1, and many show improvement over M2.2. (B) Expressionof NK1 mutants on the yeast cell surface as measured by fluorescentantibodies against a terminal epitope tag. Five NK1 mutants that hadhigh expression levels, Met-Fc binding were chosen for solubleproduction and further characterization (boxed).

FIG. 15: Aras-4 Potently Inhibits HGF-induced Met Activity. The effectof Aras-4, M.2.2D127A, and NK1 N127A on Met activation was assayed usingMadine-Darby canine kidney (MDCK) cells in the presence of variousconcentrations of HGF (20, 100, or 500 NM).

FIG. 16A-FIG. 16B: Introduction of Cysteine at the N-Terminus of VariantProteins Results in Partial Monomer/Dimer Formation. A) FPLC trace ofNi-NTA-purified cystine dimer M2.2 D127N (cdD127N), showing a mixture ofdimeric and monomeric forms. Absorbance was monitored at 280 nm. Thedimer peak was collected and used for subsequent cellular activityassays. B) SDS-PAGE of purified cdD127N and cdD127K proteins.Non-reduced NK1 proteins (left two lanes) and reduced withβ-mercaptoethanol right two lanes), supporting that the covalent dimersare the result of disulfide bonds. SDS-PAGE lane order: (L-R) Sizemarker, cdD127K (non-reduced), cdD127N (non-reduced), cdD127K (reduced),cdD127N (reduced). cdD127N: cystine dimer M2.2 D127N; cdD127K: cystinedimer M2.2 D127K.

FIG. 17: Covalently coupled NK1 Homodimers Are Potent Agonists. (top)MDCK scatter activity of the M2.2 D127N monomer (without the freecysteine residue) or the cystine dimer proteins. cdD127N: cystine dimerM2.2 D127N; cdD127K: cystine dimer M2.2 D127K. (bottom) Cystinedimerization of Aras-4 (cdAras-4) as transforms Aras-4 from anantagonist to a potent agonist.

FIG. 18A-FIG. 18B: Western Blots of Aras-4 D127C. (A) Proteins notreduced prior to addition to gel (B) Proteins reduced prior to additionto gel. Lane1 Aras-4 untreated; Lane 2 Aras-4 copper phenathrolinetreated; Aras-4 D127C untreated; Lane 4 Aras-4 D127C copperphenanthroline treated.

FIG. 19: Only an N-terminal Cysteine Mediates NK1 HomodimerizationDirectly from Yeast Cultures.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The Met receptor is expressed from a single gene product and isproteolytically processed into a 50 kD α-chain and a 140 kD β-chain. Theα-chain and first 212 residues of the β-chain comprise the Sema domain,a complex 7-bladed β-propeller fold. The remainder of the Met β-chaincomprises a cysteine-rich domain, four immunoglobulin domains, anintracellular kinase domain, and a C-terminal tail. Upon ligand bindingand dimerization, Met is cross-phosphorylated on Tyr-1234 and Tyr-1235of the intracellular kinase domain. This activity results in furtherphosphorylation of Tyr-1349 and Tyr-1356 at the C-terminus of Met, whichis a multisubstrate docking site for adapter proteins and signaltransducers Shc, Grb2, Gab1, PI-3 kinase and PLC-γ.

HGF exhibits an overall domain structure similar to coagulation factorssuch as plasminogen; it is expressed as a single-chain inactiveprecursor, which must be cleaved into its functionally active form byenzymes such as HGF activator, matriptase, hepsin, Factor XIIa, andFactor Xia. Single-chain proHGF and the cleaved two-chain HGF both bindMet with high affinity, but only two-chain HGF is capable of inducingMet activation. The HGF α-chain is comprised of an N-terminalhairpin-containing domain (PAN module; apple domain), followed by fourKringle domains (FIG. 1). The HGF β-chain consists of a serine proteasehomology domain, but lacks catalytic activity due to absence of keyresidues in the catalytic triad.

Several HGF fragments have been reported; NK1 and NK2 are naturallyoccurring HGF splice-variants, and NK4 was initially discovered throughdigestion of HGF with pancreatic elastase. These fragments are comprisedof the N-terminal and first Kringle (NK1), first and second Kringle(NK2), or first through fourth Kringle (NK4) domains. NK1 and NK2 werewere first reported to be Met antagonists, but have since beendetermined to function as weak Met agonists. In contrast, NK4 maintainsstrong binding to Met (400-600 μM), but does not induce Met activation,and thereby functions as a competitive HGF antagonist. NK1 appears tocomprise the minimal function unit of HGF, as it binds and activatesMet, albeit much more weakly than full-length HGF.

The present invention provides numerous advantages not present in knownMet binding agents. For example, in various embodiments, the engineeredpolypeptide variants of the invention bind to the Met receptor with highaffinity, possess high stability, and can be produced from microbialcultures at yields >10 mg/L. Moreover, these engineered variants alsopotently inhibit HGF-induced Met activation. Because the variants targetthe receptor directly, they are also of use in vivo and in vitromolecular imaging agents for diagnostic applications.

In an exemplary embodiment, the variants are prepared by conductingrounds of directed evolution of HGF, or a portion thereof, comprising atleast it's N-terminal and first Kringle domain (NK1), for improvementsin Met binding affinity and thermal stability.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures of analytical and synthetic organicchemistry described below are those well known and commonly employed inthe art. Standard techniques, or modifications thereof, are used forchemical syntheses and chemical analyses.

The terms “M2.1” and “M2.2” refer to variants of SEQ. ID. NO.: 1 havingthe following substitutions: (i) K62E, N127D, K137R, K170E, N193D; and(ii) K62E, Q95R, N127D, K132N, K137R, K170E, Q173R, N193D, respectively.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene. Moreover,as used herein, a nucleic acid encoding a polypeptide variant of theinvention is defined to include the nucleic acid sequence complementaryto this nucleic acid sequence.

The term “gene” means the segment of DNA involved in producing apolypeptide chain. It may include regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure. An isolated nucleic acid can be a component of anexpression vector.

Typically, isolated polypeptides of the invention have a level of puritypreferably expressed as a range. The lower end of the range of purityfor the polypeptide is about 60%, about 70% or about 80% and the upperend of the range of purity is about 70%, about 80%, about 90% or morethan about 90%.

When the polypeptides are more than about 90% pure, their purities arealso preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, mass-spectroscopy, or a similar means).

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds having a structure that is different from the generalchemical structure of an amino acid, but that functions in a mannersimilar to a naturally occurring amino acid.

“Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilic amino acids include Thr (T),Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg(R).

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg (R) and Lys (K).

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q), Ser (S) and Thr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:125-142. Exemplary hydrophobic amino acids include Ile (I), Phe (F),Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), Pro (P),and proline analogues.

“Aromatic Amino Acid” refers to a hydrophobic amino acid with a sidechain having at least one aromatic or heteroaromatic ring. The aromaticor heteroaromatic ring may contain one or more substituents such as —OH,—SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂, —NHR, —NRR, —C (0)R,—C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR and the like where each Ris independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl, (C₁-C₆)alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted(C₁-C₆) alkynyl, (C₁-C₂₁)) aryl, substituted (C₅-C₂₀) aryl, (C₆-C₂₆)alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl,substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl orsubstituted 6-26 membered alkheteroaryl. Genetically encoded aromaticamino acids include Phe (F), Tyr (Y) and Trp (W).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded apolar amino acids include Leu (L), Val (V), Ile(I), Met (M), Gly (G) and Ala (A).

“Aliphatic Amino Acid” refers to a hydrophobic amino acid having analiphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala (A), Val (V), Leu (L) and Ile (I).

The amino acid residue Cys (C) is unusual in that it can form disulfidebridges with other Cys (C) residues or other sulfonyl-containing aminoacids. The ability of Cys (C) residues (and other amino acids with —SHcontaining side chains) to exist in a peptide in either the reduced free—SH or oxidized disulfide-bridged form affects whether Cys (C) residuescontribute net hydrophobic or hydrophilic character to a peptide. WhileCys (C) exhibits a hydrophobicity of 0.29 according to the normalizedconsensus scale of Eisenberg (Eisenberg, 1984, supra), it is to beunderstood that for purposes of the present invention Cys (C) iscategorized as a polar hydrophilic amino acid, notwithstanding thegeneral classifications defined above.

The term “linker” refers to an amino-acid poly peptide spacer thatcovalently links two or more polypeptides. The linker can be 1-15 aminoacid residues. Preferably the linker is a single cysteine residue. Thelinker can also have the amino acid sequence SEQ ID NO:64KESCAKKQRQHMDS.

As will be appreciated by those of skill in the art, the above-definedcategories are not mutually exclusive. Thus, amino acids having sidechains exhibiting two or more physical-chemical properties can beincluded in multiple categories. For example, amino acid side chainshaving aromatic moieties that are further substituted with polarsubstituents, such as Tyr (Y), may exhibit both aromatic hydrophobicproperties and polar or hydrophilic properties, and can therefore beincluded in both the aromatic and polar categories. The appropriatecategorization of any amino acid will be apparent to those of skill inthe art, especially in light of the detailed disclosure provided herein.

Certain amino acid residues, called “helix breaking” amino acids, have apropensity to disrupt the structure of a-helices when contained atinternal positions within the helix. Amino acid residues exhibiting suchhelix-breaking properties are well-known in the art (see, e.g., Chou andFasman, Ann. Rev. Biochem. 47:251-276) and include Pro (P), Gly (G) andpotentially all D-amino acids (when contained in an L-peptide;conversely, L-amino acids disrupt helical structure when contained in aD-peptide) as well as a proline analogue. While these helix-breakingamino acid residues fall into the categories defined above, with theexception of Gly (G) (discussed infra), these residues should not beused to substitute amino acid residues at internal positions within thehelix—they should only be used to substitute 1-3 amino acid residues atthe N-terminus and/or C-terminus of the peptide.

While the above-defined categories have been exemplified in terms of thegenetically encoded amino acids, the amino acid substitutions need notbe, and in certain embodiments preferably are not, restricted to thegenetically encoded amino acids. Indeed, many of the preferred peptidesof formula (I) contain genetically non-encoded amino acids. Thus, inaddition to the naturally occurring genetically encoded amino acids,amino acid residues in the core peptides of formula (I) may besubstituted with naturally occurring non-encoded amino acids andsynthetic amino acids.

Certain commonly encountered amino acids which provide usefulsubstitutions for the core peptides of formula (I) include, but are notlimited to, β-alanine(β-Ala) and other omega-amino acids such as3-aminopropionic acid, 2, 3-diaminopropionic acid (Dpr), 4-aminobutyricacid and so forth; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid(Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly);ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA);t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg);cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal);4-chlorophenylalanine (Phe (4-Cl)); 2-fluorophenylalanine (Phe (2-F));3-fluorophenylalanine (Phe (3-F)); 4-fluorophenylalanine (Phe (4-F));penicillamine (Pen); 1/2/3/4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MSO);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe (pNH2));N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe)and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro),N-methylated amino acids and peptoids (N-substituted glycines). Inaddition, in some embodiments the amino acid proline in the corepeptides of formula (I) is substantiated with a proline analogue,including, but not limited to, azetidine-2-carboxylate (A2C),L-Thiazolidine-4-carboxylic Acid, cis-4-hydroxy-L-proline (CHP),3,4-dehydroproline, thioproline, and isonipecotic acid (Inp).

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)    (see, e.g., Creighton, Proteins (1984)).

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take one or more of the foregoing characteristicsinto consideration are well known to those of skill in the art andinclude, but are not limited to (original residue: exemplarysubstitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu,Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile:Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr),(Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu).Embodiments of this disclosure, therefore, consider functional orbiological equivalents of a polypeptide or protein as set forth above.In particular, embodiments of the invention provides variants havingabout 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the parentpolypeptide. In various embodiments, the invention provides variantshaving this level of identity to a portion of the parent polypeptidesequence, e.g., NK1, as defined herein. In various embodiments, thevariant has at least about 95%, 96%, 97%, 98% or 99% sequence identityto the parent polypeptide or to a portion of the parent polypeptidesequence, e.g., NK1, as defined herein.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence Therein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide or protein sequences, as determined by comparing thesequences. In the art, “identity” also refers to the degree of sequencerelatedness between polypeptides or proteins, as determined by the matchbetween strings of such sequences. “Identity” can be readily calculatedby known bioinformational methods.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds. Peptides of the presentinvention can vary in size, e.g., from two amino acids to hundreds orthousands of amino acids. A larger peptide (e.g., at least 10, at least20, at least 30 or at least 50 amino acid residues) is alternativelyreferred to as a “polypeptide” or “protein”. Additionally, unnaturalamino acids, for example, β-alanine, phenylglycine, homoarginine andhomophenylalanine are also included. Amino acids that are notgene-encoded may also be used in the present invention. Furthermore,amino acids that have been modified to include reactive groups,glycosylation sequences, polymers, therapeutic moieties, biomoleculesand the like may also be used in the invention. All of the amino acidsused in the present invention may be either the D- or L-isomer. TheL-isomer is generally preferred. In addition, other peptidomimetics arealso useful in the present invention. As used herein, “peptide” or“polypeptide” refers to both glycosylated and non-glycosylated peptidesor “polypeptides”. Also included are polypetides that are incompletelyglycosylated by a system that expresses the polypeptide. For a generalreview, see, Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINOACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, NewYork, p. 267 (1983).

In the present application, amino acid residues are numbered (typicallyin the superscript) according to their relative positions from theN-terminal amino acid (e.g., N-terminal methionine) of the polypeptide,which is numbered “1”. The N-terminal amino acid may be a methionine(M), numbered “1”. The numbers associated with each amino acid residuecan be readily adjusted to reflect the absence of N-terminal methionineif the N-terminus of the polypeptide starts without a methionine. It isunderstood that the N-terminus of an exemplary polypeptide can startwith or without a methionine. Accordingly, in instances in which anamino acid linker is added to the N-terminus of a wild-type polypeptide,the first linker amino acid adjoined to the N-terminal amino acid isnumber −1 and so forth. For example, if the linker has the amino acidsequence SEQ ID NO:64 KESCAKKQRQHMDS, with the S residue adjoined to theN-terminal amino acid of the wild-type polypeptide, then the mostN-terminal linker amino acid K would be −14, while the most C-terminallinker amino acid S would be −1. In this way, the numbering of aminoacids in the wild type polypeptide and linker bound wild typepolypeptide is preserved.

The term “parent polypeptide” refers to a wild-type polypeptide and theamino acid sequence or nucleotide sequence of the wild-type polypeptideis part of a publicly accessible protein database (e.g., EMBL NucleotideSequence Database, NCBI Entrez, ExPasy, Protein Data Bank and the like).

The term “mutant polypeptide” or “polypeptide variant” or “mutein”refers to a form of a polypeptide, wherein its amino acid sequencediffers from the amino acid sequence of its corresponding wild-type(parent) form, naturally existing form or any other parent form. Amutant polypeptide can contain one or more mutations, e.g., replacement,insertion, deletion, etc. which result in the mutant polypeptide.

The term “corresponding to a parent polypeptide” (or grammaticalvariations of this term) is used to describe a polypeptide of theinvention, wherein the amino acid sequence of the polypeptide differsfrom the amino acid sequence of the corresponding parent polypeptideonly by the presence of at least amino acid variation. Typically, theamino acid sequences of the variant polypeptide and the parentpolypeptide exhibit a high percentage of identity. In one example,“corresponding to a parent polypetide” means that the amino acidsequence of the variant polypeptide has at least about 50% identity, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95% or at least about 98% identity to the amino acidsequence of the parent polypeptide. In another example, the nucleic acidsequence that encodes the variant polypeptide has at least about 50%identity, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95% or at least about 98% identity tothe nucleic acid sequence encoding the parent polypeptide.

The term “introducing (or adding etc.) a variation into a parentpolypeptide” (or grammatical variations thereof), or “modifying a parentpolypeptide” to include a variation (or grammatical variations thereof)do not necessarily mean that the parent polypeptide is a physicalstarting material for such conversion, but rather that the parentpolypeptide provides the guiding amino acid sequence for the making of avariant polypeptide. In one example, “introducing a variant into aparent polypeptide” means that the gene for the parent polypeptide ismodified through appropriate mutations to create a nucleotide sequencethat encodes a variant polypeptide. In another example, “introducing avariant into a parent polypeptide” means that the resulting polypeptideis theoretically designed using the parent polypeptide sequence as aguide. The designed polypeptide may then be generated by chemical orother means.

As used herein “NK1” consists of the N-terminal and first Kringledomains of hepatocyte growth factor. Break points in the polypeptides ofthe present invention include amino acids 28-210 of human hepatocytegrowth factor Isoform 1 (Genbank Accession ID NP_000592). Others haveused break points of 31-210 and 32-210. An alternative human hepatocytegrowth factor isoform, Isoform 3 (Genbank Accession ID NP_00101932.1) isidentical to human HGF (hHGF) Isoform 1, except for a 5 amino aciddeletion in the first Kringle domain. hHGF Isoform 1 and Isoform 3 bothpotently activate the Met receptor and NK1 proteins derived from hHGFIsoform 1 or Isoform 3 also both bind and activate the Met receptor.Break points of 28-205, 31-205, and 32-205 for NK1 based on Isoform 3variant would be identical to break points of 28-210, 31-210, and 32-210for NK1 based on the Isoform 1 variant, with the only difference beingthe deletion of 5 amino acids from the first kringle domain (K1).

The term “library” refers to a collection of different polypeptides eachcorresponding to a common parent polypeptide. Each polypeptide speciesin the library is referred to as a member of the library. Preferably,the library of the present invention represents a collection ofpolypeptides of sufficient number and diversity to afford a populationfrom which to identify a lead polypeptide. A library includes at leasttwo different polypeptides. In one embodiment, the library includes fromabout 2 to about 100,000,000 members. In another embodiment, the libraryincludes from about 10,000 to about 100,000,000 members. In yet anotherembodiment, the library includes from about 100,000 to about 100,000,000members. In a further embodiment, the library includes from about1,000,000 to about 100,000,000 members. In another embodiment, thelibrary includes from about 10,000,000 to about 100,000,000 members. Inyet another embodiment, the library includes more than 100 members.

The members of the library may be part of a mixture or may be isolatedfrom each other. In one example, the members of the library are part ofa mixture that optionally includes other components. For example, atleast two polypeptides are present in a volume of cell-culture broth. Inanother example, the members of the library are each expressedseparately and are optionally isolated. The isolated polypeptides mayoptionally be contained in a multi-well container, in which each wellcontains a different type of polypeptide. In another example, themembers of the library are each expressed as fusions to a yeast orbacteria cell or phage or viral particle.

As used herein, the term “polymeric modifying group” is a modifyinggroup that includes at least one polymeric moiety (polymer). Thepolymeric modifying group added to a polypeptide can alter a property ofsuch polypeptide, for example, its bioavailability, biological activityor its half-life in the body. Exemplary polymers include water solubleand water insoluble polymers. A polymeric modifying group can be linearor branched and can include one or more independently selected polymericmoieties, such as poly(alkylene glycol) and derivatives thereof. In oneexample, the polymer is non-naturally occurring. In an exemplaryembodiment, the polymeric modifying group includes a water-solublepolymer, e.g., poly(ethylene glycol) and derivatived thereof (PEG,m-PEG), poly(propylene glycol) and derivatives thereof (PPG, m-PPG) andthe like. In a preferred embodiment, the poly(ethylene glycol) orpoly(propylene glycol) has a molecular weight that is essentiallyhomodisperse. In one embodiment the polymeric modifying group is not anaturally occurring polysaccharide.

The term “targeting moiety,” as used herein, refers to species that willselectively localize in a particular tissue or region of the body. Thelocalization is mediated by specific recognition of moleculardeterminants, molecular size of the targeting agent or conjugate, ionicinteractions, hydrophobic interactions and the like. Other mechanisms oftargeting an agent to a particular tissue or region are known to thoseof skill in the art. Exemplary targeting moieties include antibodies,antibody fragments, transferrin, HS-glycoprotein, coagulation factors,serum proteins, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO and the like.

The term “Fc-fusion protein”, as used herein, is meant to encompassproteins, in particular therapeutic proteins, comprising animmunoglobulin-derived moiety, which will be called herein the“Fc-moiety”, and a moiety derived from a second, non-immunoglobulinprotein, which will be called herein the “therapeutic moiety”,irrespective of whether or not treatment of disease is intended.

As used herein, “therapeutic moiety” means any agent useful for therapyincluding, but not limited to, antibiotics, anti-inflammatory agents,anti-tumor drugs, cytotoxins, and radioactive agents. “Therapeuticmoiety” includes prodrugs of bioactive agents, constructs in which morethan one therapeutic moiety is bound to a carrier, e.g, multivalentagents. Therapeutic moiety also includes proteins and constructs thatinclude proteins.

As used herein, “anti-tumor drug” means any agent useful to combatcancer including.

As used herein, “a cytotoxin or cytotoxic agent” means any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Other toxinsinclude, for example, ricin, CC-1065 and analogues, the duocarmycins.Still other toxins include diptheria toxin, and snake venom (e.g., cobravenom).

As used herein, “a radioactive agent” includes any radioisotope that iseffective in diagnosing or destroying a tumor. Examples include, but arenot limited to, indium-111, cobalt-60, fluorine-18, copper-64,copper-67, lutetium-177, or technicium-99m. Additionally, naturallyoccurring radioactive elements such as uranium, radium, and thorium,which typically represent mixtures of radioisotopes, are suitableexamples of a radioactive agent. The metal ions are typically chelatedwith an organic chelating moiety. The radioactive agent or radionuclidecan be a component of an imaging agent.

Near-infrared dyes can also be conjugated using standard chemistries foroptical imaging applications. “Near infrared” refers to radiation in theportion of the electromagnetic spectrum adjacent to that portionassociated with visible light, for example, from about 0.7 μm to about 1μm. The near infrared dye may include, for example, a cyanine orindocyanine derivative such as Cy5.5. The infrared dye may also includephosphoramidite dyes, for example, IRDye® 800 (LI-COR® Biosciences).

Many useful chelating groups, crown ethers, cryptands and the like areknown in the art and can be incorporated into the compounds of theinvention (e.g., EDTA, DTPA, DOTA, NTA, HDTA, etc. and their phosphonateanalogs such as DTPP, EDTP, HDTP, NTP, etc). See, for example, Pitt etal., “The Design of Chelating Agents for the Treatment of IronOverload,” In, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE; Martell,Ed.; American Chemical Society, Washington, D.C., 1980, pp. 279-312;Lindoy, THE CHEMISTRY OF MACROCYCLIC LIGAND COMPLEXES; CambridgeUniversity Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY;Springer-Verlag, New York, 1989, and references contained therein.Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, et al., Eds., American Chemical Society, Washington,D.C., 1982, pp. 370-387; Kasina et al., Bioconjugate Chem., 9: 108-117(1998); Song et al., Bioconjugate Chem., 8: 249-255 (1997). These metalbinding agents can be used to bind a metal ion detectable in an imagingmodality.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial, which when combined with the conjugate retains the conjugates'activity and is non-reactive with the subject's immune systems.“Pharmaceutically acceptable carrier” includes solids and liquids, suchas vehicles, diluents and solvents. Examples include, but are notlimited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Other carriers may alsoinclude sterile solutions, tablets including coated tablets andcapsules. Typically such carriers contain excipients such as starch,milk, sugar, certain types of clay, gelatin, stearic acid or saltsthereof, magnesium or calcium stearate, talc, vegetable fats or oils,gums, glycols, or other known excipients. Such carriers may also includeflavor and color additives or other ingredients. Compositions comprisingsuch carriers are formulated by well known conventional methods.

As used herein, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intrathecal, intralesional, orsubcutaneous administration, administration by inhalation, or theimplantation of a slow-release device, e.g., a mini-osmotic pump, to thesubject. Administration is by any route including parenteral andtransmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal),particularly by inhalation. Parenteral administration includes, e.g.,intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Moreover, whereinjection is to treat a tumor, e.g., induce apoptosis, administrationmay be directly to the tumor and/or into tissues surrounding the tumor.Other modes of delivery include, but are not limited to, the use ofliposomal formulations, intravenous infusion, transdermal patches, etc.

The term “ameliorating” or “ameliorate” refers to any indicia of successin the treatment of a pathology or condition, including any objective orsubjective parameter such as abatement, remission or diminishing ofsymptoms or an improvement in a patient's physical or mental well-being.Amelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination and/or apsychiatric evaluation.

The term “therapy” refers to “treating” or “treatment” of a disease orcondition including preventing the disease or condition from occurringin a subject (e.g., human) that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), providing relief from the symptoms or side-effects of thedisease (including palliative treatment), and relieving the disease(causing regression of the disease).

The term “effective amount” or “an amount effective to” or a“therapeutically effective amount” or any grammatically equivalent termmeans the amount that, when administered to an animal or human fortreating a disease, is sufficient to effect treatment for that disease.An effective amount can also refer to the amount necessary to cause acellular response, including for example, apoptosis, cell cycleinitiation, and/or signal transduction.

The term “pharmaceutically acceptable salts” includes salts of theactive compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituents found on the compoundsdescribed herein. When compounds of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., Journal of Pharmaceutical Science,66: 1-19 (1977)). Certain specific compounds of the present inventioncontain both basic and acidic functionalities that allow the compoundsto be converted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

“Reactive functional group,” as used herein refers to groups including,but not limited to, olefins, acetylenes, alcohols, phenols, ethers,oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides,cyanates, isocyanates, thiocyanates, isothiocyanates, amines,hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acidsisonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters,sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds. Reactive functional groups alsoinclude those used to prepare bioconjugates, e.g., N-hydroxysuccinimideesters, maleimides and the like. Methods to prepare each of thesefunctional groups are well known in the art and their application ormodification for a particular purpose is within the ability of one ofskill in the art (see, for example, Sandler and Karo, eds. ORGANICFUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989).

III. The Embodiments

The Variants

The present invention provides an hHGF polypeptide including at leastone amino acid in at least one position in which this amino acid is notfound in the parent hHGF polypeptide (wild type). The inventionencompasses variants of all isoforms of hHGF including, but not limitedto isoforms 1 and 3. Isoform 3 (NCBI accession NP 001010932) includesthe five amino acid deletion (SFLPS) underlined in SEQ. ID. NO.:1(isoform 1), below.

In an exemplary embodiment, the invention provides a variant of SEQ. ID.NO.: 1 having at least one amino acid substitution.

In an exemplary embodiment, the variant is an isolated variant.Furthermore, in various embodiments, the variant exhibits at least onedesirable characteristic not present in the present polypeptide.Exemplary characteristics include, but are not limited to, an increasein affinity for the Met receptor, an increase in thermal stability,increase or decrease in conformational flexibility and an increasedagonist or antagonistic activity towards the Met receptor. As will beappreciated by those of skill in the art, the variant may exhibit anycombination of two or more of these improved characteristics.

In an exemplary embodiment, the polypeptide variant is an antagonist forthe Met receptor. In various embodiments, the variant is an agonist ofthe Met receptor

In an exemplary embodiment, the invention provides an hHGF polypeptidevariant having a sequence which is a member selected from SEQ. ID.NO.:2-22.

An exemplary parent polypeptide is wild type HGF isoform 1(HGF NCBIaccession NP_000592)

(SEQ. ID NO.: 1) MWVTKLLPAL LLQHVLLHLL LLPIAIPYAE GQRKRRNTIHEFKKSAKTTL IKIDPALKIKTKKVNTADQC ANRCTRNKGLPFTCKAFVFD KARKQCLWFP FNSMSSGVKKEFGHEFDLYENKDYIRNCII GKGRSYKGTV SITKSGIKCQPWSSMIPHEH SFLPSSYRGK DLQENYCRNPRGEEGGPWCFTSNPEVRYEV CDIPQCSEVE CMTCNGESYR GLMDHTESGKICQRWDHQTPHRHKFLPERY PDKGFDDNYC RNPDGQPRPWCYTLDPHTRW EYCAIKTCAD NTMNDTDVPLETTECIQGQGEGYRGTVNTI WNGIPCQRWD SQYPHEHDMT PENFKCKDLRENYCRNPDGSESPWCFTTDP NIRVGYCSQI PNCDMSHGQDCYRGNGKNYM GNLSQTRSGL TCSMWDKNMEDLHRHIFWEPDASKLNENYC RNPDDDAHGP WCYTGNPLIP WDYCPISRCEGDTTPTIVNLDHPVISCAKT KQLRVVNGIP TRTNIGWMVSLRYRNKHICG GSLIKESWVL TARQCFPSRDLKDYEAWLGIHDVHGRGDEK CKQVLNVSQL VYGPEGSDLV LMKLARPAVLDDFVSTIDLPNYGCTIPEKT SCSVYGWGYT GLINYDGLLRVAHLYIMGNE KCSQHHRGKV TLNESEICAGAEKIGSGPCEGDYGGPLVCE QHKMRMVLGV IVPGRGCAIP NRPGIFVRVA YYAKWIHKIILTYKVPQS

In SEQ. ID. NO.: 1, the signal peptide comprises amino acids 1-31. TheN-terminal domain comprises amino acids 39-122. The Kringle 1 domaincomprises amino acids 126-207; Kringle 2 comprises amino acids 208-289;Kringle 3 comprises amino acids 302-384; Kringle 4 comprises amino acids388-470. The serine protease-like domain comprises 495-719.

In an exemplary embodiment, variants of the invention have a sequenceidentity with the parent polypeptide of at least about 80%, at leastabout 85%, at least about 90%, at least about 95% or at least about 96%,97%, 98% or 99%. In various embodiments, the variants of the inventionhave a sequence identity with the parent poly peptide of at least about99.2%, at least about 99.4%, at least about 99.6% or at least about99.8%.

In an exemplary embodiment, the positions of SEQ. ID. NO.: 1, which aremutated include one or more of 62, 64, 77, 95, 125, 127, 130, 132, 137,142, 148, 154, 170, 173 and 193. As those of skill will realize, anycombination of these positions can be mutated. In various embodiments,analogous positions of isoform 3 are mutated.

In an exemplary embodiment, an amino acid of the parent polypeptide isaltered from K to a member selected from E, N and R. In an exemplaryembodiment, an amino acid in the parent polypeptide is altered from Q toR. In an exemplary embodiment, an amino acid in the parent polypeptideis altered from I to a member selected from T and V. In an exemplaryembodiment, an amino acid of the parent polypeptide is altered from N toD. In some embodiments the D can be reverted back to N of the parentpolypeptide.

In various embodiments, the amino acid at position 42 is an F or a C. Invarious embodiments, the amino acid at position 62 is changed from K,found in the wild type parent polypeptide to E. In various embodiments,position 64 is a V or an A. In various embodiments, position 77 is an Nor an S. In various embodiments, the amino acid at position 95 is a Q,or anR. In various embodiments, the amino acid at position 125 ischanged from I, found in the wild type parent polypeptide, to T. Invarious embodiments, the amino acid at position 127 can be D, N, K, R orA. In various embodiments, the amino acid at position 130 is changedfrom I to V. In various embodiments, the amino acid at position 132 ischanged from a K, to an N or R. In various embodiments, the amino acidat position 137 is a K or an R. In various embodiments, the amino acidof position 154 is an S or an A. In various embodiments, the amino acidat position 170 is a K, or an E. In various embodiments, the amino acidat position 173 is a Q or a R. In various embodiments, the amino acid atposition 193 is a N, or a D. In various embodiments, the amino acid atposition 42 is an F or a C. In various embodiments, the amino acid atposition 96 is a C or an R. As those of skill will appreciate, anycombination of these changes, as well as any combination of those setforth in the tables that follow, can be present in a polypeptide variantof the invention.

Tables 1, 2 and 3 show exemplary mutations of the invention.

TABLE 1 N- domain Linker K1 domain 62 95 125 127 130 132 137 170 173 193hHGF K Q I N I K K K Q N Consensus E R T D V N R E R D M2.1 E D R E DM2.2 E R D N R E R D

TABLE 2 Individual sequence mutations of NK1 mutants isolated from thethird round of directed evolution. SEQ ID NO: 1 is wild-type; onlydifferences from wild-type sequence are shown in SEQ ID NOs: 2-22; blankspaces mean the wild-type hHGF residue is retained. SEQ ID

Isofm bp AA 28 30 33 37 38 42 44 48 58 62 64 65 75 77 82 95 1 Y E R N TF K T K K V N T N F Q 2 1 15 12 R E A S R 3 1 21 15 E A I R 4 1 16 14 EA S R 5 1 18 15 K G E A D S 6 1 19 15 A R E A S R 7 1 20 15 E A S R 8 116 13 E A I 9 1 28 20 D A C R E A S R 10 1 14 12 G E S R 11 1 17 15 H EA S R SED ID

96 98 101 123 125 127 130 132 135 137 142 148 154 168 170 173 181 190193 1 C W F D I N I K S K I K S R K Q R F N 2 D R A E R Y D 3 V T V N RT A E R Y D 4 D V N R E A E R Y D 5 D V N R V E R Y D 6 D V N R E E W YD 7 T V N R E A Q E R Y D 8 A D R N R E E R Y D 9 R R T V N R T A E R WD 10 D R R A E R Y D 11 T V N R T A E R Y D bp: number of base pairmutations AA: number of amino acid mutations

indicates data missing or illegible when filed

TABLE 3 Individual sequence mutations of NK1 mutants isolated from thethird round of directed evolution. SEQ ID NO: 1 is wild-type; onlydifferences from wild-type sequence are shown in SEQ ID NOs: 2-22. Isofmbp AA 30 33 46 58 62 64 65 75 77 78 79 95 101 1 E R A K K V N T N K G QF 12 1 17 13 E A S R 13 1 16 11 E A R 14 1 20 17 V E A S R V 15 1 18 13E A S R 16 1 17 13 R E A S R R 17 1 21 16 E A S R R R 18 1 16 14 E A S R19 1 14 9 D R 20 1 24 16 G R E A S R 21 1 21 15 K R E I R 22 1 14 12 G ES R 112 123 127 130 132 135 137 142 148 154 166 170 173 181 190 193 1 FD N I K S K I K S S K Q R F N 12 D N R V A E R Y D 13 D V N R E R Y D 14S D V N R T A E R Y D 15 D V N R E R W Y D 16 D N R E R Y D 17 D R V E AN E R Y D 18 D V N R E A E R Y D 19 D N R E R Y D 20 A D R N R A E R Y D21 A D R N R A E R Y D 22 D N R A E R Y D bp: number of base pairmutations AA: number of amino acid mutations

Conjugates

The present invention provides conjugates of the variants of theinvention with one or more conjugation partner. Exemplary conjugationpartners include polymers, targeting agents, therapeutic agents,cytotoxic agents, chelating agents and detectable agents. Those of skillwill recognize that there is overlap between these non-limiting agentcategories.

The conjugation partner or “modifying group” can be any conjugatablemoiety. Exemplary modifying groups are discussed below. The modifyinggroups can be selected for their ability to alter the properties (e.g.,biological or physicochemical properties) of a given polypeptide.Exemplary polypeptide properties that may be altered by the use ofmodifying groups include, but are not limited to, pharmacokinetics,pharmacodynamics, metabolic stability, biodistribution, watersolubility, lipophilicity, tissue targeting capabilities and thetherapeutic activity profile. Modifying groups are useful for themodification of polypeptides of use in diagnostic applications or in invitro biological assay systems.

In some embodiments, a HGF variant of the instant invention is combinedwith an Fc moiety. The Fc-moiety may be derived from a human or animalimmunoglobulin (Ig) that is preferably an IgG. The IgG may be an IgG1,IgG2, IgG3 or IgG4. It is also preferred that the Fc-moiety is derivedfrom the heavy chain of an immunoglobulin, preferably an IgG. Morepreferably, the Fc-moiety comprises a portion, such as e.g. a domain, ofan immunoglobulin heavy chain constant region. Such Ig constant regionpreferably comprises at least one Ig constant domain selected from anyof the hinge, CH2, CH3 domain, or any combination thereof. It ispreferred that the Fc-moiety comprises at least a CH2 and CH3 domain. Itis further preferred that the Fc-moiety comprises the IgG hinge region,the CH2 and the CH3 domain.

Fc domains of the IgG1 subclass are often used as the Fc moiety, becauseIgG1 has the longest serum half-life of any of the serum proteins.Lengthy serum half-life can be a desirable protein characteristic foranimal studies and potential human therapeutic use. In addition, theIgG1 subclass possesses the strongest ability to carry out antibodymediated effector functions.

The primary effector function that may be most useful in a fusionprotein is the ability for an IgG1 antibody to mediate antibodydependent cellular cytotoxicity. On the other hand, this could be anundesirable function for a fusion protein that functions primarily as anantagonist. Several of the specific amino acid residues that areimportant for antibody constant region-mediated activity in the IgG1subclass have been identified. Inclusion or exclusion of these specificamino acids therefore allows for inclusion or exclusion of specificimmunoglobulin constant region-mediated activity.

In accordance with the present invention, the Fc-moiety may also bemodified in order to modulate effector functions. For instance, thefollowing Fc mutations, according to EU index positions (Kabat et al.,1991), can be introduced if the Fc-moiety is derived from IgG1:T250Q/M428L; M252Y/S254T/T256E+H433K/N434F;E233P/L234V/L235A/AΔ236+A327G/A330S/P331S; E333A; K322A.

Further Fc mutations may e.g. be the substitutions at EU index positionsselected from 330, 331 234, or 235, or combinations thereof. An aminoacid substitution at EU index position 297 located in the CH2 domain mayalso be introduced into the Fc-moiety in the context of the presentinvention, eliminating a potential site of N-linked carbohydrateattachment. The cysteine residue at EU index position 220 may also bereplaced.

The Fc-fusion protein of the invention may be a monomer or dimer. TheFc-fusion protein may also be a “pseudo-dimer”, containing a dimericFc-moiety (e.g. a dimer of two disulfide-bridged hinge-CH2-CH3constructs), of which only one is fused to a therapeutic moiety.

The Fc-fusion protein may be a heterodimer, containing two differenttherapeutic moieties, or a homodimer, containing two copies of a singletherapeutic moiety.

In some embodiments, the in vivo half-life of the HGF variants can beenhanced with polyethylene glycol (PEG) moieties. Chemical modificationof polypeptides with PEG (PEGylation) increases their molecular size andtypically decreases surface- and functional group-accessibility, each ofwhich are dependent on the number and size of the PEG moieties attachedto the polypeptide. Frequently, this modification results in animprovement of plasma half-live and in proteolytic-stability, as well asa decrease in immunogenicity and hepatic uptake (Chaffee et al. J. Clin.Invest. 89: 1643-1651 (1992); Pyatak et al. Res. Commun. Chem. PatholPharmacol. 29: 113-127 (1980)). For example, PEGylation of interleukin-2has been reported to increase its antitumor potency in vivo (Katre etal. Proc. Natl. Acad. Sci. USA. 84: 1487-1491 (1987)) and PEGylation ofa F(ab′)2 derived from the monoclonal antibody A7 has improved its tumorlocalization (Kitamura et al. Biochem. Biophys. Res. Commun. 28:1387-1394 (1990)). Thus, in another embodiment, the in vivo half-life ofa polypeptide derivatized with a PEG moiety by a method of the inventionis increased relative to the in vivo half-life of the non-derivatizedparent polypeptide.

The increase in polypeptide in vivo half-life is best expressed as arange of percent increase relative to the parent polypeptide. The lowerend of the range of percent increase is about 40%, about 60%, about 80%,about 100%, about 150% or about 200%. The upper end of the range isabout 60%, about 80%, about 100%, about 150%, or more than about 250%.

Many water-soluble polymers are known to those of skill in the art andare useful in practicing the present invention. The term water-solublepolymer encompasses species such as saccharides (e.g., dextran, amylose,hyalouronic acid, poly(sialic acid), heparans, heparins, etc.);poly(amino acids), e.g., poly(aspartic acid) and poly(glutamic acid);nucleic acids; synthetic polymers (e.g., poly(acrylic acid),poly(ethers), e.g., poly(ethylene glycol); peptides, proteins, and thelike. The present invention may be practiced with any water-solublepolymer with the sole limitation that the polymer must include a pointat which the remainder of the conjugate can be attached. See, forexample, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten,Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb.Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky,Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie,57:5-29 (2002).

In another embodiment, analogous to those discussed above, the modifiedsugars include a water-insoluble polymer, rather than a water-solublepolymer. The conjugates of the invention may also include one or morewater-insoluble polymers. This embodiment of the invention isillustrated by the use of the conjugate as a vehicle with which todeliver a therapeutic polypeptide in a controlled manner. Polymeric drugdelivery systems are known in the art. See, for example, Dunn et al.,Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol. 469, American Chemical Society, Washington, D.C. 1991. Those ofskill in the art will appreciate that substantially any known drugdelivery system is applicable to the conjugates of the presentinvention.

Representative water-insoluble polymers include, but are not limited to,polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics andpolyvinylphenol and copolymers thereof.

Representative biodegradable polymers of use in the conjugates of theinvention include, but are not limited to, polylactides, polyglycolidesand copolymers thereof, poly(ethylene terephthalate), poly(butyricacid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, pluronics and the like.

Exemplary resorbable polymers include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., JBiomed Mater. Res. 22: 993-1009 (1988).

Polymers that are components of hydrogels are also useful in the presentinvention. Hydrogels are polymeric materials that are capable ofabsorbing relatively large quantities of water. Examples of hydrogelforming compounds include, but are not limited to, polyacrylic acids,sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine,gelatin, carrageenan and other polysaccharides,hydroxyethylenemethacrylic acid (HEMA), as well as derivatives thereof,and the like. Hydrogels can be produced that are stable, biodegradableand bioresorbable. Moreover, hydrogel compositions can include subunitsthat exhibit one or more of these properties.

In another embodiment, the gel is a thermoreversible gel.Thermoreversible gels including components, such as pluronics, collagen,gelatin, hyalouronic acid, polysaccharides, polyurethane hydrogel,polyurethane-urea hydrogel and combinations thereof are presentlypreferred.

In yet another exemplary embodiment, the conjugate of the inventionincludes a component of a liposome. Liposomes can be prepared accordingto methods known to those skilled in the art, for example, as describedin Eppstein et al., U.S. Pat. No. 4,522,811, which issued on Jun. 11,1985. For example, liposome formulations may be prepared by dissolvingappropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine,stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, andcholesterol) in an inorganic solvent that is then evaporated, leavingbehind a thin film of dried lipid on the surface of the container. Anaqueous solution of the active compound or its pharmaceuticallyacceptable salt is then introduced into the container. The container isthen swirled by hand to free lipid material from the sides of thecontainer and to disperse lipid aggregates, thereby forming theliposomal suspension.

The present invention also provides conjugates analogous to thosedescribed above in which the polypeptide is conjugated to a therapeuticmoiety, diagnostic moiety, targeting moiety, toxin moiety or the like.Each of the above-recited moieties can be a small molecule, naturalpolymer (e.g., polypeptide) or a synthetic polymer.

In various embodiments, the variant is conjugated to a component of amatrix for tissue regeneration. Exemplary matrices are known in the artand it is within the ability of a skilled worker to select and modify anappropriate matrix with an HGF variant of the invention. The HGFvariants of the invention are generally of use in regenerative medicineapplications, including the regeneration of, e.g., liver, muscle, nerveand cardiac tissue. See, for example, Isobe et al., Hepatocyte growthfactor: Effects on immune-mediated heart diseases. Trends CardiovascMed., 16 (6) pp. 188-93 (2006); Anderson et al., The role ofcytoprotective cytokines in cardiac ischemia/reperfusion injury. Journalof Surgical Research, 148 (2) pp. 164-71 (2008); Maulik et al., Growthfactors and cell therapy in myocardial regeneration. Journal ofMolecular and Cellular Cardiology, 44 (2) pp. 219-27 (2008); Chen etal., In vivo hepatocyte growth factor gene transfer reduces myocardialischemia-reperfusion injury through its multiple actions. JournalCardiac Failure, 13 (10) pp. 874-83 (2007); Kondo et al., Treatment ofacute myocardial infarction by hepatocyte growth factor gene transfer:The first demonstration of myocardial transfer of a “functional” geneusing ultrasonic microbubble destruction. Journal of the AmericanCollege of Cardiology, 44 (3) pp. 644-53 (2004); Schirmer et al.,Stimulation of collateral artery growth: travelling further down theroad to clinical application. Heart, 95 (3) pp. 191-197 (2009); Zhu etal., Transplantation of adipose-derived stem cells overexpressing hHGFinto cardiac tissue. Biochemical and Biophysical Research Communication,379 (4) pp. 1084-90 (2009); Carlsson et al., Quantitative MRmeasurements of regional and global left ventricular function and strainafter intramyocardial transfer of VM202 into infarcted swine myocardium.Am. J. Physiol. Heart Circ. Physiol., 295 (2) pp. H522-32 (2008);Schuldiner et al., Effects of eight growth factors on thedifferentiation of cells derived from human embryonic cells. PNAS, 97(21) pp. 11307-11312 (2000); Cassano et al., Magic-Factor 1, a PartialAgonist of Met, Induces Muscle Hypertrophy by Protecting MyogenicProgenitors from Apoptosis. PloS One vol. 3 (9) pp. 1-13 (2008); Linkeet al., Stems cells in the dog heart are self-renewing clonogenic, andmultipotent and regenerate infarcted myocardium, improving cardiacfunction. PNAS vol. 102 (25) pp. 8966-8971 (2005); Takahara et al.,Metron Factor-1 Prevents Liver Injury without Promoting Tumor Growth andMetastasis, Hepatology, 47 (6) pp. 2010-2025 (2008); Neuss et al.,Functional Expression of HGF and HGF Receptor/c-met in Adult HumanMesenchymal Stem Cells Suggest a Role in Cell Mobilization, TissueRepair, and Wound Healing. Stem Cells, 22 pp. 405-414 (2004).

In a still further embodiment, the invention provides conjugates thatlocalize selectively in a particular tissue due to the presence of atargeting agent as a component of the conjugate. In an exemplaryembodiment, the targeting agent is a protein. Exemplary proteins includetransferrin (brain, blood pool), HS-glycoprotein (bone, brain, bloodpool), antibodies (brain, tissue with antibody-specific antigen, bloodpool), coagulation factors V-XII (damaged tissue, clots, cancer, bloodpool), serum proteins, e.g., α-acid glycoprotein, fetuin, α-fetalprotein (brain, blood pool), β2-glycoprotein (liver, atherosclerosisplaques, brain, blood pool), G-CSF, GM-CSF, M-CSF, and EPO (immunestimulation, cancers, blood pool, red blood cell overproduction,neuroprotection), albumin (increase in half-life), IL-2 and IFN-α.

In another embodiment, the invention provides a conjugate between apolypeptide of the invention and a therapeutic moiety. Therapeuticmoieties, which are useful in practicing the instant invention includedrugs from a broad range of drug classes having a variety ofpharmacological activities. Methods of conjugating therapeutic anddiagnostic agents to various other species are well known to those ofskill in the art. See, for example Hermanson, BIOCONJUGATE TECHNIQUES,Academic Press, San Diego, 1996; and Dunn et al., Eds. POLYMERIC DRUGSAND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol. 469, AmericanChemical Society, Washington, D.C. 1991.

Classes of useful therapeutic moieties include, for example,antineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, β-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine). Also included within this class areradioisotope-based agents for both diagnosis and therapy, and conjugatedtoxins, such as ricin, geldanamycin, mytansin, CC-1065, theduocarmycins, Chlicheamycin and related structures and analoguesthereof.

The therapeutic moiety can also be a hormone (e.g., medroxyprogesterone,estradiol, leuprolide, megestrol, octreotide or somatostatin); endocrinemodulating drugs (e.g., contraceptives (e.g., ethinodiol, ethinylestradiol, norethindrone, mestranol, desogestrel, medroxyprogesterone).Of use in various embodiments of the invention are conjugates withestrogens (e.g., diethylstilbesterol), glucocorticoids (e.g.,triamcinolone, betamethasone, etc.) and progestogens, such asnorethindrone, ethynodiol, norethindrone, levonorgestrel; thyroid agents(e.g., liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful modifying groups include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H2 antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

In various embodiments, the conjugate is formed by reaction between areactive amino acid and a reactive conjugation partner for the reactiveamino acid. Both the reactive amino acid and the reactive conjugationpartner include within their framework one or more reactive functionalgroup. One of the two binding species may include a “leaving group” (oractivating group) refers to those moieties, which are easily displacedin enzyme-regulated nucleophilic substitution reactions oralternatively, are replaced in a chemical reaction utilizing anucleophilic reaction partner (e.g., an amino acid moiety carrying asufhydryl group). It is within the abilities of a skilled person toselect a suitable leaving group for each type of reaction. Manyactivated sugars are known in the art. See, for example, Vocadlo et al.,In CARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol. 2, Ernst et al. Ed.,Wiley-VCH Verlag: Weinheim, Germany, 2000; Kodama et al., TetrahedronLett. 34: 6419 (1993); Lougheed, et al., J. Biol. Chem. 274: 37717(1999)).

In various embodiments, the amino acid substitution, which is thevariant (or a variant) of naturally occurring HGF, is the locus forattachment of the conjugation partner, e.g., a side-chain amino acid,e.g., cysteine, lysine, serine, etc.

Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive sugar moieties are those, which proceed under relativelymild conditions. These include, but are not limited to nucleophilicsubstitutions (e.g., reactions of amines and alcohols with acyl halides,active esters), electrophilic substitutions (e.g., enamine reactions)and additions to carbon-carbon and carbon-heteroatom multiple bonds(e.g., Michael reaction, Diels-Alder addition). These and other usefulreactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

Reactive Functional Groups

Useful reactive functional groups on a reactive amino acid or reactiveconjugation partner include, but are not limited to:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters;    -   (b) hydroxyl groups, which can be converted to, e.g., esters,        ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the functional group of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc; and    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive sugar nucleus or modifying group. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

The group linking the polypeptide and conjugation partner can also be across-linking group, e.g., a zero- or higher-order cross-linking group(for reviews of crosslinking reagents and crosslinking procedures see:Wold, F., Meth. Enzymol. 25: 623-651, 1972; Weetall, H. H., and Cooney,D. A., In: ENZYMES AS DRUGS. (Holcenberg, and Roberts, eds.) pp.395-442, Wiley, New York, 1981; Ji, T. H., Meth. Enzymol. 91: 580-609,1983; Mattson et al., Mol. Biol. Rep. 17: 167-183, 1993, all of whichare incorporated herein by reference). Preferred crosslinking reagentsare derived from various zero-length, homo-bifunctional, andhetero-bifunctional crosslinking reagents. Zero-length crosslinkingreagents include direct conjugation of two intrinsic chemical groupswith no introduction of extrinsic material. Agents that catalyzeformation of a disulfide bond belong to this category. Another exampleis reagents that induce condensation of a carboxyl and a primary aminogroup to form an amide bond such as carbodiimides, ethylchloroformate,Woodward's reagent K (2-ethyl-5-phenylisoxazolium-3′-sulfonate), andcarbonyldiimidazole. In addition to these chemical reagents, the enzymetransglutaminase (glutamyl-peptide γ-glutamyltransferase; EC 2.3.2.13)may be used as zero-length crosslinking reagent. This enzyme catalyzesacyl transfer reactions at carboxamide groups of protein-boundglutaminyl residues, usually with a primary amino group as substrate.Preferred homo- and hetero-bifunctional reagents contain two identicalor two dissimilar sites, respectively, which may be reactive for amino,sulfhydryl, guanidino, indole, or nonspecific groups.

Exemplary conjugation partners attached to the polypeptides of theinvention include, but are not limited to, PEG derivatives (e.g.,alkyl-PEG, acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG,aryl-PEG), PPG derivatives (e.g., alkyl-PPG, acyl-PPG, acyl-alkyl-PPG,alkyl-acyl-PPG carbamoyl-PPG, aryl-PPG), therapeutic moieties,diagnostic moieties, mannose-6-phosphate, heparin, heparan, Slex,mannose, mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF, proteins,chondroitin, keratan, dermatan, albumin, integrins, antennaryoligosaccharides, peptides and the like.

In addition to covalent attachments, the polypeptides of the instantinvention can be attached onto the surface of a biomaterial throughnon-covalent interactions. Non covalent protein incorporation can bedone, for example, through encapsulation or absorption. Attachment ofthe polypeptides of the instant invention to a biomaterial may bemediated through heparin. In some embodiments, the polypeptides of theinstant invention are attached to a heparin-alginate polymer andalginate as described in Harada et al., J. Clin. Invest. (1994)94:623-630; Laham et al., Circulation (1999) 1865-1871 and referencescited therein. In other embodiments, the polypeptides of the instantinvention are attached to a collagen based biomaterial.

Imaging Agents

An exemplary conjugate of the invention is an imaging agent comprising avariant of the invention and a detectable moiety, which is detectable inan imaging modality. There is a critical need for molecular imagingprobes that will specifically target Met receptors in living subjectsand allow noninvasive characterization of tumors for patient-specificcancer treatment and disease management. The ability to detectMet-expressing tumors through non-invasive imaging could also serve asan indicator of metastatic risk.

Exemplary imaging modalities in which the conjugates of the inventionfind use include, without limitation, positron emission tomography (PET)in which a variant of the invention is tagged with a positron emittingisotope. Typical isotopes include ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu, ⁶²Cu, ¹²⁴I,⁷⁶Br ⁸²Rb and ⁶⁸Ga, with ¹⁸F being the most clinically utilized. Thevariants can also be incorporated into ultrasound agents, magneticresonance imaging agents, X-ray agents, CT agents, gamma camerascintigraphy agents and fluorescent imaging agents. Additionaldetectable moieties and methods of imaging are set forth in the Methodssection hereinbelow.

Compared to other tumor targets, such as integrin receptors, thedevelopment of Met-based imaging agents has been extremely limited. Thusfar, a few Met-specific monoclonal antibodies have been radiolabeled andused to image Met-expressing tumors in mouse models. One limitation ofthis approach is that intact antibodies must be allowed to clear fromthe body for several days after injection before imaging studies can beperformed or high background signals will result; this limits theradioisotopes that can be used for imaging to those with long half-lives(e.g., ¹²⁵I). Moreover, the antibodies in these imaging studies wereMet-receptor agonists, which could potentially induce Met receptoractivation. To address these issues, a Met-binding Fab antibody fragmentand a Met-binding peptide were both identified from phage-displayedlibraries. Radiolabeled versions of these Met-targeting agents wereshown to image tumors in living subjects; however tumor uptake was low,possibly due to the weak Met receptor binding affinity of these probes.These studies highlight that there is substantial room for improvementand further probe development.

In an exemplary embodiment, the conjugation partner is attached to apolypeptide variant of the invention via a linkage that is cleaved underselected conditions. Exemplary conditions include, but are not limitedto, a selected pH (e.g., stomach, intestine, endocytotic vacuole), thepresence of an active enzyme (e.g, esterase, reductase, oxidase), light,heat and the like. Many cleavable groups are known in the art. See, forexample, Jung et al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshiet al., J. Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J.Immunol., 124: 913-920 (1980); Bouizar et al., Eur. J. Biochem., 155:141-147 (1986); Park et al., J. Biol. Chem., 261: 205-210 (1986);Browning et al., J. Immunol., 143: 1859-1867 (1989).

Pharmaceutical Compositions

Polypeptides and their conjugates of the invention have a broad range ofpharmaceutical applications.

Thus, in another aspect, the invention provides a pharmaceuticalcomposition including at least one polypeptide or polypeptide conjugateof the invention and a pharmaceutically acceptable diluent, carrier,vehicle, additive or combinations thereof. Pharmaceutical compositionsof the invention are suitable for use in a variety of drug deliverysystems. Suitable formulations for use in the present invention arefound in Remington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985). For a brief review of methods fordrug delivery, see, Langer, Science 249:1527-1533 (1990).

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablematrices, such as microspheres (e.g., polylactate polyglycolate), mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administeredsubcutaneously or parenterally, e.g., intravenously. Thus, the inventionprovides compositions for parenteral administration, which include thecompound dissolved or suspended in an acceptable carrier, preferably anaqueous carrier, e.g., water, buffered water, saline, PBS and the like.The compositions may also contain detergents such as Tween 20 and Tween80; stabilizers such as mannitol, sorbitol, sucrose, and trehalose; andpreservatives such as EDTA and meta-cresol. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8.

In some embodiments the glycopeptides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid-derivatized glycopeptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion, which isfirmly embedded and anchored in the membrane. It must also have areactive portion, which is chemically available on the aqueous surfaceof the liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate, which is added later. In some cases it ispossible to attach the target agent to the connector molecule directly,but in most instances it is more suitable to use a third molecule to actas a chemical bridge, thus linking the connector molecule which is inthe membrane with the target agent or carbohydrate which is extended,three dimensionally, off of the vesicle surface.

The compounds prepared by the methods of the invention may also find useas diagnostic reagents. For example, labeled compounds can be used tolocate areas of inflammation or tumor metastasis in a patient suspectedof having an inflammation. For this use, the compounds can be labeledwith ¹²⁵I, ¹⁴C, or tritium.

Antibodies and Nucleic Acids

In various embodiments, the invention provides an isolated nucleic acidencoding a polypeptide variant according to any of the embodiments setforth hereinabove. In various embodiments, the invention provides anucleic acid complementary to this nucleic acid.

In various embodiments, the invention provides an expression vectorincluding a nucleic acid encoding a polypeptide variant according to anyof the embodiments set forth hereinabove operatively linked to apromoter.

In various embodiments, the invention provides an antibody capable ofspecifically binding to a polypeptide variant of the invention. Alsoprovided is an isolated nucleic acid encoding this antibody, anexpression system encoding this antibody in which the nucleic acidencoding the antibody is operatively linked to a promoter. A cellexpressing the antibody is also provided. In various embodiments, theinvention provides an expression vector including a nucleic acidencoding a polypeptide variant according to any of the embodiments setforth hereinabove.

Libraries

Also provided in various embodiments is a library of variant hHGFpolypeptides comprising a plurality of different members, wherein eachmember of the library corresponds to a common parent polypeptide hHGFand wherein each member of the library comprises an amino acid at aposition at which the amino acid is not found in the parent polypeptide.

IV. Methods

Chemical Synthesis

Polyeptide variants of the invention may be prepared using conventionalstep-wise solution or solid phase synthesis (see, e.g., ChemicalApproaches to the Synthesis of Peptides and Proteins, Williams et al.,Eds., 1997, CRC Press, Boca Raton Fla., and references cited therein;Solid Phase Peptide Synthesis: A Practical Approach, Atherton &Sheppard, Eds., 1989, IRL Press, Oxford, England, and references citedtherein).

Alternatively, the peptides of the invention may be prepared by way ofsegment condensation, as described, for example, in Liu et al., 1996,Tetrahedron Lett. 37(7)933 936; Baca, et al., 1995, J. Am. Chem. Soc.117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein Res.45:209-216; Schnölzer and Kent, 1992, Science 256:221-225; Liu and Tam,1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam, 1994, Proc.Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li, 1988, Int. J.Peptide Protein Res. 31:322-334). Segment condensation is a particularlyuseful method for synthesizing embodiments containing internal glycineresidues. Other methods useful for synthesizing the peptides of theinvention are described in Nakagawa et al., 1985, J. Am. Chem. Soc.107:7087-7092.

Polypeptide variants containing N- and/or C-terminal blocking groups canbe prepared using standard techniques of organic chemistry. For example,methods for acylating the N-terminus of a peptide or amidating oresterifying the C-terminus of a peptide are well-known in the art. Modesof carrying other modifications at the N- and/or C-terminus will beapparent to those of skill in the art, as will modes of protecting anyside-chain functionalities as may be necessary to attach terminalblocking groups. Pharmaceutically acceptable salts (counter ions) can beconveniently prepared by ion-exchange chromatography or other methods asare well known in the art.

Compounds of the invention which are in the form of tandem multimers canbe conveniently synthesized by adding the linker(s) to the peptide chainat the appropriate step in the synthesis. Alternatively, the helicalsegments can be synthesized and each segment reacted with the linker. Ofcourse, the actual method of synthesis will depend on the composition ofthe linker. Suitable protecting schemes and chemistries are well known,and will be apparent to those of skill in the art.

Compounds of the invention which are in the form of branched networkscan be conveniently synthesized using the trimeric and tetrameric resinsand chemistries described in Tam, 1988, Proc. Natl. Acad. Sci. USA85:5409-5413 and Demoor et al., 1996, Eur. J. Biochem. 239:74-84.Modifying the synthetic resins and strategies to synthesize branchednetworks of higher or lower order, or which contain combinations ofdifferent core peptide helical segments, is well within the capabilitiesof those of skill in the art of peptide chemistry and/or organicchemistry. Formation of disulfide linkages, if desired, is generallyconducted in the presence of mild oxidizing agents.

Chemical oxidizing agents may be used, or the compounds may simply beexposed to atmospheric oxygen to effect these linkages. Various methodsare known in the art, including those described, for example, by Tam etal., 1979, Synthesis 955-957; Stewart et al., 1984, Solid Phase PeptideSynthesis, 2d Ed., Pierce Chemical Company Rockford, Ill.; Ahmed et al.,1975, J. Biol. Chem. 250:8477-8482; and Pennington et al., 1991 Peptides1990 164-166, Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands. Anadditional alternative is described by Kamber et al., 1980, Helv. Chim.Acta 63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res. 26:92-97. Any of thesemethods may be used to form disulfide linkages in the peptides of theinvention.

Acquisition of Polypeptide Coding Sequences

General Recombinant Technology

The creation of mutant polypeptides, which incorporate an O-linkedglycosylation sequence of the invention can be accomplished by alteringthe amino acid sequence of a corresponding parent polypeptide, by eithermutation or by full chemical synthesis of the polypeptide. Thepolypeptide amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA sequence encoding thepolypeptide at preselected bases to generate codons that will translateinto the desired amino acids. The DNA mutation(s) are preferably madeusing methods known in the art.

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook and Russell, Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Ausubel et al., eds., Current Protocols inMolecular Biology (1994).

Nucleic acid sizes are given in either kilobases (kb) or base pairs(bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage & Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984). Entire genescan also be chemically synthesized. Purification of oligonucleotides isperformed using any art-recognized strategy, e.g., native acrylamide gelelectrophoresis or anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255: 137-149 (1983).

The sequence of the cloned wild-type polypeptide genes, polynucleotideencoding mutant polypeptides, and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16: 21-26(1981).

In an exemplary embodiment, the glycosylation sequence is added byshuffling polynucleotides. Polynucleotides encoding a candidatepolypeptide can be modulated with DNA shuffling protocols. DNA shufflingis a process of recursive recombination and mutation, performed byrandom fragmentation of a pool of related genes, followed by reassemblyof the fragments by a polymerase chain reaction-like process. See, e.g.,Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751 (1994); Stemmer,Nature 370:389-391 (1994); and U.S. Pat. Nos. 5,605,793, 5,837,458,5,830,721 and 5,811,238.

Cloning and Subcloning of a Wild-Type Peptide Coding Sequence

Numerous polynucleotide sequences encoding wild-type polypeptides havebeen determined and are available from a commercial supplier, e.g.,human growth hormone, e.g., GenBank Accession Nos. NM 000515, NM 002059,NM 022556, NM 022557, NM 022558, NM 022559, NM 022560, NM 022561, and NM022562.

The rapid progress in the studies of human genome has made possible acloning approach where a human DNA sequence database can be searched forany gene segment that has a certain percentage of sequence homology to aknown nucleotide sequence, such as one encoding a previously identifiedpolypeptide. Any DNA sequence so identified can be subsequently obtainedby chemical synthesis and/or a polymerase chain reaction (PCR) techniquesuch as overlap extension method. For a short sequence, completely denovo synthesis may be sufficient; whereas further isolation of fulllength coding sequence from a human cDNA or genomic library using asynthetic probe may be necessary to obtain a larger gene.

Alternatively, a nucleic acid sequence encoding a polypeptide can beisolated from a human cDNA or genomic DNA library using standard cloningtechniques such as polymerase chain reaction (PCR), where homology-basedprimers can often be derived from a known nucleic acid sequence encodinga polypeptide. Most commonly used techniques for this purpose aredescribed in standard texts, e.g., Sambrook and Russell, supra.

cDNA libraries suitable for obtaining a coding sequence for a wild-typepolypeptide may be commercially available or can be constructed. Thegeneral methods of isolating mRNA, making cDNA by reverse transcription,ligating cDNA into a recombinant vector, transfecting into a recombinanthost for propagation, screening, and cloning are well known (see, e.g.,Gubler and Hoffman, Gene, 25: 263-269 (1983); Ausubel et al., supra).Upon obtaining an amplified segment of nucleotide sequence by PCR, thesegment can be further used as a probe to isolate the full-lengthpolynucleotide sequence encoding the wild-type polypeptide from the cDNAlibrary. A general description of appropriate procedures can be found inSambrook and Russell, supra.

A similar procedure can be followed to obtain a full length sequenceencoding a wild-type polypeptide, e.g., any one of the GenBank AccessionNos mentioned above, from a human genomic library. Human genomiclibraries are commercially available or can be constructed according tovarious art-recognized methods. In general, to construct a genomiclibrary, the DNA is first extracted from an tissue where a polypeptideis likely found. The DNA is then either mechanically sheared orenzymatically digested to yield fragments of about 12-20 kb in length.The fragments are subsequently separated by gradient centrifugation frompolynucleotide fragments of undesired sizes and are inserted inbacteriophage λ vectors. These vectors and phages are packaged in vitro.Recombinant phages are analyzed by plaque hybridization as described inBenton and Davis, Science, 196: 180-182 (1977). Colony hybridization iscarried out as described by Grunstein et al., Proc. Natl. Acad. Sci.USA, 72: 3961-3965 (1975).

Based on sequence homology, degenerate oligonucleotides can be designedas primer sets and PCR can be performed under suitable conditions (see,e.g., White et al., PCR Protocols: Current Methods and Applications,1993; Griffin and Griffin, PCR Technology, CRC Press Inc. 1994) toamplify a segment of nucleotide sequence from a cDNA or genomic library.Using the amplified segment as a probe, the full-length nucleic acidencoding a wild-type polypeptide is obtained.

Upon acquiring a nucleic acid sequence encoding a wild-type polypeptide,the coding sequence can be subcloned into a vector, for instance, anexpression vector, so that a recombinant wild-type polypeptide can beproduced from the resulting construct. Further modifications to thewild-type polypeptide coding sequence, e.g., nucleotide substitutions,may be subsequently made to alter the characteristics of the molecule.

Introducing Mutations into a Polypeptide Sequence

From an encoding polynucleotide sequence, the amino acid sequence of awild-type polypeptide can be determined. Subsequently, this amino acidsequence may be modified to alter the protein's glycosylation pattern,by introducing additional glycosylation sequence(s) at various locationsin the amino acid sequence.

A variety of mutation-generating protocols are established and describedin the art. See, e.g., Zhang et al., Proc. Natl. Acad. Sci. USA, 94:4504-4509 (1997); and Stemmer, Nature, 370: 389-391 (1994). Theprocedures can be used separately or in combination to produce variantsof a set of nucleic acids, and hence variants of encoded polypeptides.Kits for mutagenesis, library construction, and otherdiversity-generating methods are commercially available.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Botstein and Shortie, Science, 229: 1193-1201(1985)), mutagenesis using uracil-containing templates (Kunkel, Proc.Natl. Acad. Sci. USA, 82: 488-492 (1985)), oligonucleotide-directedmutagenesis (Zoller and Smith, Nucl. Acids Res., 10: 6487-6500 (1982)),phosphorothioate-modified DNA mutagenesis (Taylor et al., Nucl. AcidsRes., 13: 8749-8764 and 8765-8787 (1985)), and mutagenesis using gappedduplex DNA (Kramer et al., Nucl. Acids Res., 12: 9441-9456 (1984)).

Other methods for generating mutations include point mismatch repair(Kramer et al., Cell, 38: 879-887 (1984)), mutagenesis usingrepair-deficient host strains (Carter et al., Nucl. Acids Res., 13:4431-4443 (1985)), deletion mutagenesis (Eghtedarzadeh and Henikoff,Nucl. Acids Res., 14: 5115 (1986)), restriction-selection andrestriction-purification (Wells et al., Phil. Trans. R. Soc. Lond. A,317: 415-423 (1986)), mutagenesis by total gene synthesis (Nambiar etal., Science, 223: 1299-1301 (1984)), double-strand break repair(Mandecki, Proc. Natl. Acad. Sci. USA, 83: 7177-7181 (1986)),mutagenesis by polynucleotide chain termination methods (U.S. Pat. No.5,965,408), and error-prone PCR (Leung et al., Biotechniques, 1: 11-15(1989)).

Modification of Nucleic Acids for Preferred Codon Usage in a HostOrganism

The polynucleotide sequence encoding a polypeptide variant can befurther altered to coincide with the preferred codon usage of aparticular host. For example, the preferred codon usage of one strain ofbacterial cells can be used to derive a polynucleotide that encodes apolypeptide variant of the invention and includes the codons favored bythis strain. The frequency of preferred codon usage exhibited by a hostcell can be calculated by averaging frequency of preferred codon usagein a large number of genes expressed by the host cell (e.g., calculationservice is available from web site of the Kazusa DNA Research Institute,Japan). This analysis is preferably limited to genes that are highlyexpressed by the host cell. U.S. Pat. No. 5,824,864, for example,provides the frequency of codon usage by highly expressed genesexhibited by dicotyledonous plants and monocotyledonous plants.

At the completion of modification, the polpeptide variant codingsequences are verified by sequencing and are then subcloned into anappropriate expression vector for recombinant production in the samemanner as the wild-type polypeptides.

Expression of Mutant Polypeptides

Following sequence verification, the polypeptide variant of the presentinvention can be produced using routine techniques in the field ofrecombinant genetics, relying on the polynucleotide sequences encodingthe polypeptide disclosed herein.

Expression Systems

To obtain high-level expression of a nucleic acid encoding a mutantpolypeptide of the present invention, one typically subclones apolynucleotide encoding the mutant polypeptide into an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator and a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook and Russell, supra, and Ausubelet al., supra. Bacterial expression systems for expressing the wild-typeor mutant polypeptide are available in, e.g., E. coli, Bacillus sp.,Salmonella, and Caulobacter. Kits for such expression systems arecommercially available. Eukaryotic expression systems for mammaliancells, yeast, and insect cells are well known in the art and are alsocommercially available. In one embodiment, the eukaryotic expressionvector is an adenoviral vector, an adeno-associated vector, or aretroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically includes atranscription unit or expression cassette that contains all theadditional elements required for the expression of the mutantpolypeptide in host cells. A typical expression cassette thus contains apromoter operably linked to the nucleic acid sequence encoding themutant polypeptide and signals required for efficient polyadenylation ofthe transcript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding the polypeptide is typically linked to acleavable signal peptide sequence to promote secretion of thepolypeptide by the transformed cell. Such signal peptides include, amongothers, the signal peptides from tissue plasminogen activator, insulin,and neuron growth factor, and juvenile hormone esterase of Heliothisvirescens. Additional elements of the cassette may include enhancersand, if genomic DNA is used as the structural gene, introns withfunctional splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322-based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

In some exemplary embodiments the expression vector is chosen frompCWin1, pCWin2, pCWin2/MBP, pCWin2-MBP-SBD (pMS39), andpCWin2-MBP-MCS-SBD (pMXS39) as disclosed in co-owned U.S. patentapplication filed Apr. 9, 2004 which is incorporated herein byreference.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as abaculovirus vector in insect cells, with a polynucleotide sequenceencoding the mutant polypeptide under the direction of the polyhedrinpromoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

When periplasmic expression of a recombinant protein (e.g., a hgh mutantof the present invention) is desired, the expression vector furthercomprises a sequence encoding a secretion signal, such as the E. coliOppA (Periplasmic Oligopeptide Binding Protein) secretion signal or amodified version thereof, which is directly connected to 5′ of thecoding sequence of the protein to be expressed. This signal sequencedirects the recombinant protein produced in cytoplasm through the cellmembrane into the periplasmic space. The expression vector may furthercomprise a coding sequence for signal peptidase 1, which is capable ofenzymatically cleaving the signal sequence when the recombinant proteinis entering the periplasmic space. More detailed description forperiplasmic production of a recombinant protein can be found in, e.g.,Gray et al., Gene 39: 247-254 (1985), U.S. Pat. Nos. 6,160,089 and6,436,674.

As discussed above, a person skilled in the art will recognize thatvarious conservative substitutions can be made to any wild-type ormutant polypeptide or its coding sequence while still retaining thebiological activity of the polypeptide. Moreover, modifications of apolynucleotide coding sequence may also be made to accommodate preferredcodon usage in a particular expression host without altering theresulting amino acid sequence.

Transfection Methods

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of the mutantpolypeptide, which are then purified using standard techniques (see,e.g., Colley et al., J. Biol. Chem. 264: 17619-17622 (1989); Guide toProtein Purification, in Methods in Enzymology, vol. 182 (Deutscher,ed., 1990)). Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss, Methods inEnzymology 101: 347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA, or other foreign genetic material into a host cell (see,e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe mutant polypeptide.

Detection of Expression of Mutant Polypeptides in Host Cells

After the expression vector is introduced into appropriate host cells,the transfected cells are cultured under conditions favoring expressionof the mutant polypeptide. The cells are then screened for theexpression of the recombinant polypeptide, which is subsequentlyrecovered from the culture using standard techniques (see, e.g., Scopes,Protein Purification: Principles and Practice (1982); U.S. Pat. No.4,673,641; Ausubel et al., supra; and Sambrook and Russell, supra).

Several general methods for screening gene expression are well knownamong those skilled in the art. First, gene expression can be detectedat the nucleic acid level. A variety of methods of specific DNA and RNAmeasurement using nucleic acid hybridization techniques are commonlyused (e.g., Sambrook and Russell, supra). Some methods involve anelectrophoretic separation (e.g., Southern blot for detecting DNA andNorthern blot for detecting RNA), but detection of DNA or RNA can becarried out without electrophoresis as well (such as by dot blot). Thepresence of nucleic acid encoding a mutant polypeptide in transfectedcells can also be detected by PCR or RT-PCR using sequence-specificprimers.

Second, gene expression can be detected at the polypeptide level.Various immunological assays are routinely used by those skilled in theart to measure the level of a gene product, particularly usingpolyclonal or monoclonal antibodies that react specifically with amutant polypeptide of the present invention (e.g., Harlow and Lane,Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988;Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniquesrequire antibody preparation by selecting antibodies with highspecificity against the mutant polypeptide or an antigenic portionthereof. The methods of raising polyclonal and monoclonal antibodies arewell established and their descriptions can be found in the literature,see, e.g., Harlow and Lane, supra; Kohler and Milstein, Eur. J.Immunol., 6: 511-519 (1976). More detailed descriptions of preparingantibody against the mutant polypeptide of the present invention andconducting immunological assays detecting the mutant polypeptide areprovided in a later section.

Purification of Recombinantly Produced Mutant Polypeptides

Once the expression of a recombinant mutant polypeptide in transfectedhost cells is confirmed, the host cells are then cultured in anappropriate scale for the purpose of purifying the recombinantpolypeptide.

1. Purification from Bacteria

When the mutant polypeptides of the present invention are producedrecombinantly by transformed bacteria in large amounts, typically afterpromoter induction, although expression can be constitutive, theproteins may form insoluble aggregates. There are several protocols thatare suitable for purification of protein inclusion bodies. For example,purification of aggregate proteins (hereinafter referred to as inclusionbodies) typically involves the extraction, separation and/orpurification of inclusion bodies by disruption of bacterial cells, e.g.,by incubation in a buffer of about 100-150 μg/mllysozyme and 0.1%Nonidet P40, a non-ionic detergent. The cell suspension can be groundusing a Polytron grinder (Brinkman Instruments, Westbury, N.Y.).Alternatively, the cells can be sonicated on ice. Alternate methods oflysing bacteria are described in Ausubel et al. and Sambrook andRussell, both supra, and will be apparent to those of skill in the art.

For further description of purifying recombinant polypeptides frombacterial inclusion body, see, e.g., Patra et al., Protein Expressionand Purification 18: 182-190 (2000).

The recombinant proteins present in the supernatant can be separatedfrom the host proteins by standard separation techniques well known tothose of skill in the art.

Immunoassays for Detection of Mutant Polypeptide Expression

To confirm the production of a recombinant mutant polypeptide,immunological assays may be useful to detect in a sample the expressionof the polypeptide. Immunological assays are also useful for quantifyingthe expression level of the recombinant hormone. Antibodies against amutant polypeptide are necessary for carrying out these immunologicalassays.

Production of Antibodies against Mutant Polypeptides

Methods for producing polyclonal and monoclonal antibodies that reactspecifically with an immunogen of interest are known to those of skillin the art (see, e.g., Coligan, Current Protocols in ImmunologyWiley/Greene, N Y, 1991; Harlow and Lane, Antibodies: A LaboratoryManual Cold Spring Harbor Press, N Y, 1989; Stites et al. (eds.) Basicand Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding, Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y., 1986;and Kohler and Milstein Nature 256: 495-497, 1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors (see, Huse et al.,Science 246: 1275-1281, 1989; and Ward et al., Nature 341: 544-546,1989).

In order to produce antisera containing antibodies with desiredspecificity, the polypeptide of interest (e.g., a mutant polypeptide ofthe present invention) or an antigenic fragment thereof can be used toimmunize suitable animals, e.g., mice, rabbits, or primates. A standardadjuvant, such as Freund's adjuvant, can be used in accordance with astandard immunization protocol. Alternatively, a synthetic antigenicpeptide derived from that particular polypeptide can be conjugated to acarrier protein and subsequently used as an immunogen.

The animal's immune response to the immunogen preparation is monitoredby taking test bleeds and determining the titer of reactivity to theantigen of interest. When appropriately high titers of antibody to theantigen are obtained, blood is collected from the animal and antiseraare prepared. Further fractionation of the antisera to enrich antibodiesspecifically reactive to the antigen and purification of the antibodiescan be performed subsequently, see, Harlow and Lane, supra, and thegeneral descriptions of protein purification provided above.

Monoclonal antibodies are obtained using various techniques familiar tothose of skill in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol.6:511-519, 1976). Alternative methods of immortalization include, e.g.,transformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and the yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host.

Additionally, monoclonal antibodies may also be recombinantly producedupon identification of nucleic acid sequences encoding an antibody withdesired specificity or a binding fragment of such antibody by screeninga human B cell cDNA library according to the general protocol outlinedby Huse et al., supra. The general principles and methods of recombinantpolypeptide production discussed above are applicable for antibodyproduction by recombinant methods.

When desired, antibodies capable of specifically recognizing a mutantpolypeptide of the present invention can be tested for theircross-reactivity against the wild-type polypeptide and thusdistinguished from the antibodies against the wild-type protein. Forinstance, antisera obtained from an animal immunized with a mutantpolypeptide can be run through a column on which a wild-type polypeptideis immobilized. The portion of the antisera that passes through thecolumn recognizes only the mutant polypeptide and not the wild-typepolypeptide. Similarly, monoclonal antibodies against a mutantpolypeptide can also be screened for their exclusivity in recognizingonly the mutant but not the wild-type polypeptide.

Polyclonal or monoclonal antibodies that specifically recognize only themutant polypeptide of the present invention but not the wild-typepolypeptide are useful for isolating the mutant protein from thewild-type protein, for example, by incubating a sample with a mutantpeptide-specific polyclonal or monoclonal antibody immobilized on asolid support.

Methods of Treatment and Diagnosis

In various embodiments, the invention provides a method of preventing,ameliorating or treating a disease state, which can be treated byinhibiting Met. In these embodiments, the invention provides a methodthat comprises administering to a subject in need thereof an amount of apolypeptide variant Met inhibitor of the invention sufficient toprevent, ameliorate or treat the disease state. An exemplary diseasestate is cancer. The disclosed agonist variants can be useful for thepromotion of cell growth, particularly for angiogenesis, and thetreatment of cardiovascular, hepatic, musculoskeletal and neuronaldiseases.

For example, in the adult, the HGF-Met pathway is involved in muscleregeneration following injury. Thus, the disclosed variants can find usein repairing muscle injuries, including for example, cardiac tissueregeneration following infaraction.

The disclosed variants can be used, for example, be used to treat orprevent liver failure or disease caused by conditions including viralinfection (such as by infection with a hepatitis virus, e.g. HAV, HBV orHCV), or other acute viral hepatitis, autoimmune chronic hepatitis,acute fatty liver of pregnancy, Budd-Chiari syndrome and veno-occlusivedisease, hyperthermia, hypoxia, malignant infiltration, Reye's syndrome,sepsis, Wilson's disease and in transplant rejection.

The disclosed variants can be used to treat or prevent acute liverfailure or disease induced by toxins, including a toxin selected frommushroom poisoning (e.g. Amanita phalloides), arsenic, carbontetrachloride (or other chlorinated hydrocarbons), copper, ethanol,iron, methotrexate and phosphorus. A particular use of the polypeptidesof the invention is in the treatment or prevention of liver damagecaused by intoxication by N-acetyl-p-aminophenol (known commercially asparacetamol or acetaminophen).

Further, the disclosed variants can be useful in the treatment followingkidney failure, supporting kidney maintenance and regeneration.

Because the polypeptide variants of the invention neutralize theactivity of HGF, they can be used in various therapeutic applications.For example, certain polypeptide variants of the invention are useful inthe prevention or treatment of hyperproliferative diseases or disorders,e.g., various forms of cancer.

In an exemplary embodiment, the invention provides a method of treatingcancer in a subject in need of such treatment. The method includesadministering to the subject a therapeutically effective amount of apolypeptide variant of the invention.

It is contemplated that the polypeptide variants of the invention can beused in the treatment of a variety of HGF responsive disorders,including, for example, HGF responsive tumor cells in lung cancer,breast cancer, colon cancer, prostate cancer, ovarian cancer, head andneck cancer, ovarian cancer, multiple myeloma, liver cancer, gastriccancer, esophageal cancer, kidney cancer, nasopharangeal cancer,pancreatic cancer, mesothelioma, melanoma and glioblastoma.

In exemplary embodiments, the cancer is a carcinoma, e.g., colorectal,squamous cell, hepatocellular, renal, breast or lung.

The polypeptide variants can be used to inhibit or reduce theproliferation of tumor cells. In such an approach, the tumor cells areexposed to a therapeutically effective amount of the polypeptide variantso as to inhibit or reduce proliferation of the tumor cell. In certainembodiments, the polypeptide variants inhibit tumor cell proliferationby at least 50%, 60%, 70%, 80%, 90%, 95% or 100%.

In certain embodiments, the polypeptide variant is used to inhibit orreduce proliferation of a tumor cell wherein the variant reduces theability of hHGF to bind to c-Met. In other embodiments, the polypeptidevariant is used to inhibit or reduce the proliferation of a tumor celleven when the polypeptide variant binds c-Met but does not substantiallyinhibit hHGF binding to c-Met.

In addition, the polypeptide variant can be used to inhibit, or slowdown tumor growth or development in a mammal. In such a method, aneffective amount of the polypeptide variant is administered to themammal so as to inhibit or slow down tumor growth in the mammal.Accordingly, the polypeptide variants can be used to treat tumors, forexample, in a mammal. The method comprises administering to the mammal atherapeutically effective amount of the polypeptide variant. Thepolypeptide variant can be administered alone or in combination withanother pharmaceutically active molecule, so as to treat the tumor.

Generally, a therapeutically effective amount of polypeptide variantwill be in the range of from about 0.1 mg/kg to about 100 mg/kg,optionally from about 1 mg/kg to about 100 mg/kg, optionally from about1 mg/kg to 10 mg/kg. The amount administered will depend on variablessuch as the type and extent of disease or indication to be treated, theoverall health status of the particular patient, the relative biologicalefficacy of the polypeptide variant delivered, the formulation of thepolypeptide variant, the presence and types of excipients in theformulation, and the route of administration. The initial dosageadministered may be increased beyond the upper level in order to rapidlyachieve the desired blood-level or tissue level, or the initial dosagemay be smaller than the optimum and the daily dosage may beprogressively increased during the course of treatment depending on theparticular situation. Human dosage can be optimized, e.g., in aconventional Phase I dose escalation study designed to run from 0.5mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors suchas route of administration, dosage amount and the disease conditionbeing treated. Exemplary dosing frequencies are once per day, once perweek and once every two weeks. A preferred route of administration isparenteral, e.g., intravenous infusion. Formulation of protein-baseddrugs is within ordinary skill in the art. In some embodiments of theinvention, the polypeptide variant, e.g., protein-based, is lyophilizedand reconstituted in buffered saline at the time of administration.

The polypeptide variants may be administered either alone or incombination with other pharmaceutically active ingredients. The otheractive ingredients, e.g., immunomodulators, can be administered togetherwith the polypeptide variant, or can be administered before or after thepolypeptide variant.

Formulations containing the polypeptide variants for therapeutic use,typically include the polypeptide variants combined with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means buffers, carriers, and excipients, that are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The carrier(s) shouldbe “acceptable” in the sense of being compatible with the otheringredients of the formulations and not deleterious to the recipient.Pharmaceutically acceptable carriers, in this regard, are intended toinclude any and all buffers, solvents, dispersion media, coatings,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is known in the art.

The formulations can be conveniently presented in a dosage unit form andcan be prepared by any suitable method, including any of the methodswell known in the pharmacy art. Remington's Pharmaceutical Sciences,18th ed. (Mack Publishing Company, 1990).

In exemplary embodiments, the polypeptide variants are used fordiagnostic purposes, either in vitro or in vivo, the polypeptidevariants typically are labeled either directly or indirectly with adetectable moiety. The detectable moiety can be any moiety which iscapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a radioisotope, suchas ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent or chemiluminescentcompound, such as fluorescein isothiocyanate, Cy5.5 (GE Healthcare),Alexa Fluro® dyes (Invitrogen), IRDye® infrared dyes (LI-COR®Biosciences), rhodamine, or luciferin; an enzyme, such as alkalinephosphatase, beta-galactosidase, or horseradish peroxidase; a spinprobe, such as a spin label; or a colored particle, for example, a latexor gold particle. It is understood that the polypeptide variant can beconjugated to the detectable moiety using a number of approaches knownin the art, for example, as described in Hunter et al. (1962) Nature144: 945; David et al. (1974) Biochemistry 13: 1014; Pain et al. (1981)J. Immunol Meth 40: 219; and Nygren (1982) J. Histochem and Cytochem.30: 407. The labels may be detected, e.g., visually or with the aid of aspectrophotometer or other detector or other appropriate imaging system.

The polypeptide variants can be employed in a wide range of immunoassaytechniques available in the art. Exemplary immunoassays include, forexample, sandwich immunoassays, competitive immunoassays,immunohistochemical procedures.

In a sandwich immunoassay, two antibodies that bind an analyte orantigen of interest are used, e.g., one immobilized onto a solidsupport, and one free in solution and labeled with a detectable moiety.When a sample containing the antigen is introduced into this system, theantigen binds to both the immobilized antibody and the labeled antibody,to form a “sandwich” immune complex on the surface of the support. Thecomplexed protein is detected by washing away non-bound samplecomponents and excess labeled antibody, and measuring the amount oflabeled antibody complexed to protein on the support's surface.Alternatively, the antibody free in solution can be detected by a thirdantibody labeled with a detectable moiety which binds the free antibody.A detailed review of immunological assay design, theory and protocolscan be found in numerous texts, including Butt, ed., (1984) PracticalImmunology, Marcel Dekker, New York; Harlow et al. eds. (1988)Antibodies, A Laboratory Approach, Cold Spring Harbor Laboratory; andDiamandis et al., eds. (1996) Immunoassay, Academic Press, Boston.

It is contemplated that the labeled polypeptide variants are useful asin vivo imaging agents, whereby the polypeptide variants can target theimaging agents to particular tissues of interest in the recipient. Aremotely detectable moiety for in vivo imaging includes the radioactiveatom ⁹⁹mTc, a gamma emitter with a half-life of about six hours.Non-limiting examples of radionuclide diagnostic agents include, for¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y,⁸⁹Zr, ⁹⁴mTc, ⁹⁴Tc, ⁹⁹mTc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴-¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ⁵²mMn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ⁸²mRb,⁸³Sr, or other γ-, β-, or positron-emitters.

Non-radioactive moieties also useful in in vivo imaging includenitroxide spin labels as well as lanthanide and transition metal ionsall of which induce proton relaxation in situ. In addition to imagingthe complexed radioactive moieties may be used in standardradioimmunotherapy protocols to destroy the targeted cell.

A wide variety of fluorescent labels are known in the art, including butnot limited to fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.Chemiluminescent labels of use may include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt or anoxalate ester.

The disclosed polypeptide variants may also be labeled with afluorescent marker so as to allow detection in vivo. In someembodiments, the fluorescent label is Cy5.5 (GE Healthcare). In otherembodiments, the fluorescent lable is an Alexa Fluro® dye (Invitrogen).In some embodiments, the fluorescent lable is an IRDye® infrared dye(LI-COR® Biosciences).

Exemplary nucleotides for high dose radiotherapy include the radioactiveatoms ⁹⁰Yt, ¹³¹I and ¹¹¹In. The polypeptide variant can be labeled with¹³¹I, ¹¹¹In and ⁹⁹mTC using coupling techniques known in the imagingarts. Similarly, procedures for preparing and administering the imagingagent as well as capturing and processing images are well known in theimaging art and so are not discussed in detail herein. Similarly,methods for performing antibody-based immunotherapies are well known inthe art. See, for example, U.S. Pat. No. 5,534,254.

EXAMPLES

The following examples are provided by way of illustration only and arenot meant to limit the scope of the invention. Those of skill in the artwill readily recognize a variety of non-critical parameters that couldbe changed or modified to yield essentially similar results.

Example 1

1.1 Protein engineering of NK1 through yeast surface display. Yeastsurface display is a powerful directed evolution technology that hasbeen used to engineer proteins for enhanced binding affinity, properfolding, and improved stability. Combinatorial libraries of NK1 proteinswere displayed on the surface of the yeast strain Saccharomycescerevesiae through genetic fusion to the yeast mating agglutinin proteinAga2p. Aga2p is disulfide bonded to Aga1p, which is covalently linked tothe yeast cell wall. In contrast to most yeast display studies, theconstruct we used here tethered the displayed NK1 proteins to theN-terminus of Aga2p (FIG. 2). It was found for this ligand-receptorsystem that this orientation reduced steric constraints of receptor andantibody labeling described below. The NK1 proteins were flanked byN-terminal hemagglutinin (HA) and C-terminal c-myc epitope tags, whichwere used to confirm expression of the construct on the yeast cellsurface and to quantitate surface expression levels. A flexible(Gly₄Ser)₃ linker at the C-terminus of the displayed NK1 protein wasused to project the protein away from the yeast cell surface to furtherminimize steric constraints.

Libraries of 10⁷-10⁸ transformants were routinely created for proteinengineering studies, with each yeast cell displaying thousands ofidentical copies of a particular NK1 mutant on its surface.High-throughput screening of tens of millions of yeast-displayed NK1mutants using fluorescent-activated cell sorting (FACS) allowed for theisolation of protein variants with desired properties, in this caseenhanced Met receptor binding affinity and/or enhanced expression. Forthis purpose, yeast-displayed NK1 libraries were stained with bothfluorescently-labeled Met-Fc fusion protein and primary and secondaryantibodies against the HA epitope tag (FIG. 2B). The use of multicolorflow cytometry enabled simultaneous and independent monitoring of bothrelative surface expression levels and Met binding by detectingphycoerythrin and Alexa-488 fluorescence, respectively. Yeast cells thatbound the highest levels of Met and possessed the highest NK1 expressionlevels were isolated. Previously, a strong correlation has been shownbetween expression levels on the yeast cell surface, and thermalstability and soluble expression yields. The sorted yeast werepropagated in culture, and the screening process was repeated severaltimes to obtain an enriched yeast population consisting of a smallnumber of unique clones.

1.2 Overview: Directed evolution of NK1 for high affinity and stabilityusing yeast surface display. An NK1 fragment was engineered for 1)enhanced thermal stability and 2) high binding affinity to Met. A firstround of directed evolution consisted largely of evolving NK1 forfunctional expression on the yeast cell surface and for modestimprovements in Met binding affinity. Pooled products were furthermutated and subjected to a second round of directed evolution in whichthey were screened independently for either improved Met bindingaffinity or enhanced stability. A third round of directed evolution wasthen conducted by performing DNA shuffling on pooled products from thesecond round, followed by screening simultaneously for improved Metbinding affinity and enhanced stability (FIG. 3).

1.3 Wild-type NK1 is not functionally expressed on the yeast cellsurface. HGF exists in two main isoforms, Isoform 1 (I1: Genbankaccession no. NP_000592) and Isoform 3 (I3: Genbank accession no.NP_001010932;

(NP_00101932) SEQ ID NO: 63 MWVTKLLPALLLQHVLLHLL LLPIAIPYAE GQRKRRNTIHEFKKSAKTTL IKIDPALKIKTKKVNTADQC ANRCTRNKGLPFTCKAFVFDKARKQCLWFP FNSMSSGVKK EFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSYRGKDLQENYCRNPRGEEGGPWCFTSNPE VRYEVCDIPQCSEVECMTCN GESYRGLMDH TESGKICQRW DHQTPHRHKLPERYPDKGF DDNYCRNPDG QPRPWCYTLD PHTRWEYCAIKTCADNTMND TDVPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPH EHDMTPENFK CKDLRENYCR NPDGSESPWCFTTDPNIRVG YCSQIPNCDM SHGQDCYRGNGKNYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLPNYGCTIPEKTSCSVY GWGYTGLINY DGLLRVAHLY IMGNEKCSQH HRGKVTLNESEICAGAEKIG SGPCEGDYGG PLVCEQHKMR MVLGVIVPGRGCAIPNRPGI FVRVAYYAKW IHKIILTYKV PQSHGF I1 and I3 are identical in sequence, except for a 5 amino aciddeletion in the first Kringle domain (K1) of I3. Yeast display plasmid,pTMY-HA, was used to express NK1 or NK1 I3 on the yeast cell surface asa genetic fusion to the yeast cell wall protein Aga2p (FIG. 2). Similarresults were found for both NK1 I1 and NK1 I3. Yeast-displayed NK1 wasstained for relative expression (through antibody detection of the HAtag) and binding to 20 or 200 nM of Met-Fc (R&D Systems) labeled withAlexa 488. Since heparin is required for the wild-type NK1-Metinteraction, this experiment was conducted both in the presence (FIG.4A, top) and absence (FIG. 4A, bottom) of 2 μM heparin (Lovenox,Sanofi-Aventis). Flow cytometry was used to detect yeast expressing NK 1I1 on the yeast cell surface. Only low levels of binding to solubleMet-Fc was observed (FIG. 4, x-axis vs. y-axis). Binding levels areshown after heating yeast-displayed NK1 to 70° C. (FIG. 4B). As shownbelow, soluble NK1 I1 produced from the yeast Pichia pastoris iscompletely unfolded at 60° C. (FIG. 4). Collectively, this datademonstrates that yeast-displayed wild-type NK1 is not functionallyexpressed on the yeast cell surface.

1.4 Engineering NK1 for improved affinity and stability using yeastsurface display. Three separate rounds of directed evolution were usedto evolve NK1 for improvements in stability and Met binding affinitycompared to wild-type NK1. Since NK1 was not functionally expressed onthe yeast cell surface, the first round of directed evolution largelyconsisted of screening yeast-displayed NK1 mutants to isolate clonesthat bound to the Met receptor. Towards this goal, we generated alibrary of approximately 3×10⁷ NK1 mutants by error-prone PCR usingnucleotide analogs 8-oxo-dGTP and dPTP (TriLink BioTechnologies). Asneither NK1 I1 nor NK1 I3 are functionally expressed on the yeast cellsurface, it was not clear which isoform would be most amenable toaffinity maturation through directed evolution. Therefore, we used equalamounts of NK1 I1 and NK1 I3 as starting templates to generate acombined NK1 mutant library based on both I1 and 13. Sequencing ofrandom clones from the yeast-displayed library confirmed equalrepresentation of NK1 I1 and NK1 I3.

Yeast prefer to grow at 30° C., however, they often show improvedexpression of more complex proteins at 20° C. Therefore, two rounds oflibrary sorting were conducted after inducing protein expression on theyeast cell surface at 20° C. to enable improved folding of NK1 mutants,and FACS was used to isolated yeast cells that exhibited detectablebinding to 200 nM Alexa-488 labeled Met-Fc (Met-Fc A488) (FIG. 5A).Subsequent library sorts were conducted in parallel using either 20° C.or 30° C. induction temperatures with the goal of screening for mutantswith improved stability using the 30° C. expression temperature. Afterfive rounds of sorting with each strategy (5 rounds using 20° C.expression temperature, or 2 rounds with 20° C. followed by 3 roundswith 30° C. expression temperature) the library clearly containedmembers that bound to 200 nM Met-Fc.

For a second round of directed evolution pooled mutants from the finalsorts of the first round of directed evolution were randomly mutated byerror-prone PCR to generate a library of approximately 8×10⁷ uniquemutants. The first two rounds of sorting of this library were conductedusing a 20° C. expression temperature to first recover mutants thatbound to soluble Met-Fc A488. For subsequent rounds, we sorted inparallel either for improvements in expression (i.e. folding stability),which has been shown to correlate to improved thermal stability, or forimprovements in Met binding affinity (FIG. 5B). Expression at elevatedtemperatures (37° C.) was used to impart sorting stringency for improvedstability, while improved binding to decreasing concentrations ofsoluble Met-Fc A488 was used for affinity sorting stringency.

Finally, a third round of directed evolution consisted of DNA shufflingof the final pools of the stability- and affinity-enhanced mutants fromthe second round of directed evolution to generate a third generationlibrary of approximately 2×10⁷ unique transformants. This library wassimultaneously screened for both enhanced stability (via high cellsurface expression level upon 37° C. induction) and enhanced affinity(through improved binding to substantially decreasing concentrations ofMet-Fc A488). The first, second and third rounds of sorting used 40, 20and 2 nM Met-Fc A488, respectively. After three rounds of sorting, theresulting pool of mutants expressed well at 37° C. and bound strongly to2 nM Met-Fc A488 (FIG. 6, middle). Subsequent sorts were conducted bylabeling with 2 nM Met-Fc A488, followed by an unbinding step in thepresence of excess unlabeled competitor, in this case recombinant HGF(R&D Systems). Clones that retained Met binding after 24 hr in thepresence of excess HGF competitor were isolated by FACS. This processwas repeated until a pool of NK1 mutants that retained binding to Met-FcA488 following a 2 day unbinding step in the presence of excess HGF as aMet-Fc competitor (FIG. 6, right).

A pool of NK1 variants was identified in which the variants areefficiently expressed on the yeast cell surface at elevated temperaturesand maintain persistent binding to 2 nM soluble Met even after a 2 dayunbinding step in the presence of excess HGF competitor (FIG. 6).

1.5 Sequence analysis of affinity and stability-enhanced NK1 mutants. Inparallel to performing Round 3 of directed evolution, characterizationbegan of promising mutants from Round 2. Eight random mutants weresequenced from each of the final two sort rounds for each sortingstrategy (20° C. affinity sort strategy, and 37° C. stability sortstrategy). Interestingly, all 32 clones sequenced were based on NK1 I1,even though sequencing of the initial library indicated relatively equalproportions of NK1 Isoform 1 and Isoform 3. Additionally, a number offavored or consensus mutations were evident. 10 mutations repeatedlyappeared in clones randomly sequenced from the library sort products,and eight of these mutations were present in over half of the randomlyselected clones. These dominant mutations are highlighted in bold inTable 1. Due to the wide variety of mutations, none of the individualclones contained all eight of these mutations. However, one clonecontained five of the eight most frequent mutations (K62E, N127D, K137R,K170E, N193D; this clone is termed M2.1). The remaining three mutations(Q95R, K132N, Q173R) were added onto the background of this clone togenerate the NK1 mutant we termed M2.2. Further sequence analysis ofthese mutations highlighted a number of interesting observations, whichare further discussed below.

The sort products from the two strategies did not produce many of theexact same clones, but did however exhibit a remarkable overlap inconsensus sequences. The negative correlation between I125T and N127Dobserved in the M2 (second round directed evolution) products persistedwith the M3 (third round directed evolution) products. Of the 30sequenced clones, 25 contained the N127D mutation, none of which alsocontained the I125T mutation. However, each of the five clones notcontaining N127D did contain the I125T mutation. K62E/V64A and1130V/K132N consensus mutations occurred with only a 2 amino acidspacing.

All of the eight consensus mutations from M2 products were present inthe M3 products (recall M2.2=K62E, Q95R, N127D, K132N, K137R, K170E,Q173R, N193D). There were five additional consensus mutations that arosein over 50% of the M3 products: V64A, N77S, 1130V, S154A, and F190Y.

TABLE 4 Sequence Sustitutions Present in Certain Variants ProteinMutations Activity NK1 None (wild-type NK1) Agonist M2.2 K62E, Q95R,N127D, K132N, Weak K137R, K170E, Q173R, N193D agonist M2.2 K62E, Q95R,K132N, K137R, Agonist D127N K170E, Q173R, N193D (an N127D mutation inM2.2 was reverted back to the wild-type ‘N’. M2.2 K62E, Q95R, N127A,K132N, Antagonist D127A K137R, K170E, Q173R, N193D M2.2 K62E, Q95R,N127K, K132N, Antagonist D127K K137R, K170E, Q173R, N193D M2.2 K62E,Q95R, N127R, K132N, Antagonist D127R K137R, K170E, Q173R, N193D Aras-4M3S7.2.11 R33G, K58R, K62E, V64A, N77S, Antagonist Q95R, D123A, N127D,K132R, S135N, K137R, S154A, K170E, Q173R, F190Y, N193D

Example 2

2.1 Soluble production of wild-type NK1 and NK1 mutants in the yeaststrain P. pastoris. Briefly, DNA encoding for wild-type NK1, M2.1, orM2.2 containing an N-terminal FLAG epitope tag (DYKDDDDK) and aC-terminal hexahistidine tag were cloned into the secretion plasmidpPIC9K. Constructs were transformed into P. pastoris, and were selectedfor growth on YPD-agar plates containing 4 mg/mL Geneticin and screenedfor NK1 expression by Western blotting of culture supernatant. FIG. 7Ashows that M2.1 and M2.2 express well at 30° C., while wild-type NK1expresses at much lower levels. This data is in agreement with previousstudies that report engineering for enhanced protein stability usingyeast-surface display also confers improved recombinant expressionlevels. However, reducing the expression temperature to 20° C. enabledefficient expression of wild-type NK1 (data not shown). NK1 and mutantexpression were scaled up to 0.5 L in shake flask cultures and purifiedusing immobilized nickel affinity chromatography followed by gelfiltration on a Superdex™ 75 column (GE Healthcare). Several milligramsof mutants M2.1 and M2.2 were obtained from one 0.5 L shake flask,without any optimization, indicating that even higher yields could beobtained by modifying induction conditions or through fermentation.

2.2 Mutants M2.1 and M2.2 exhibit higher thermal stability thanwild-type NK1. To test thermal stability, M2.1 and M2.2 were expressedon the yeast cell surface, heated to varying temperatures, and theretention of binding to fluorescently labeled Met-Fc was measured byflow cytometry (FIG. 8A). NK1 mutants M2.1 and M2.2 have T_(m) values onthe surface of yeast of 61.0±1.4° C. and 61.4±0.7° C., respectively. Itwas not possible to monitor stability of yeast-displayed wild-type NK1since it was not functionally expressed on the yeast cell surface.

To test the stability of soluble proteins, secondary structure unfoldingof purified, soluble mutants was monitored using circular dichroism (CD)on a Jasco J-815 CD spectrometer. CD scans of the mutant proteinsidentified a peak at 208 nm, owing largely to the β-sheet structuralelement. The CD scans of M2.1 and M2.2 resembled that of wild-type NK1,illustrating the mutant proteins contain the same overall secondarystructural elements as wild-type NK1 (FIG. 8B). A CD spectra ofwild-type NK1 at 80° C. resembles that of a random coil, demonstratingthe ability to monitor the unfolding of secondary structural elementsusing circular dichroism (FIG. 8B) Using this information, the unfoldingof this secondary structure was monitored by variable temperature CDscans (FIG. 8C). In each of these assays, the M2.1 and M2.2 exhibitedhigher thermal stability (63.6±0.3° C. and 67.8±0.2° C., respectively)compared to wild-type NK1 (T_(m)=50.9±0.2° C.). To further confirm theseresults, the melting of a local maxima at 236 nm to that of a randomcoil for M2.1 was monitored. The same T_(m) was observed for melting at208 nm. A summary of thermal stability (T_(m)) of wild-type and mutantNK1 proteins as determined by CD temperature melts is shown in Table 5.

TABLE 5 Tm ± std. dev. (° C.) NK1 50.7 ± 0.2 NK1 N127A 47.9 ± 0.7 M2.163.9 ± 0.5 M2.2 69.0 ± 1   M2.2 D127N 65.5 ± 0.5 M2.2 D127A 63.7 ± 0.1M2.2 D127K 62.5 ± 0.1 M2.2 D127R 62.3 ± 0.5

2.3 The Effects of Salt concentration on protein stability. To retainits structural integrity, it was observed that wild-type NK1 must bemaintained in buffer containing high salt concentrations (>200-300 mMNaCl). As further evidence of this requirement, wild-type NK1 exhibiteda broad, delayed elution profile on size exclusion chromatography withbuffer containing moderate salt concentration (137 mM) (FIG. 9 andinset), suggesting unfolding and/or non-specific binding to the columnunder these conditions. In contrast, M2.1 and M2.2 eluted as a single,sharp peak on size exclusion chromatography under similar moderate saltconditions (FIG. 9).

Example 3

3.1 Point mutations at the NK1 homodimerization interface Residue N127lies within the linker region connecting the N and K1 domains (FIG. 1).The side chain of this asparagine residue forms two hydrogen bonds. TheN127D variant was frequently observed among the library-isolatedvariants. (Tables 2 and 3). To probe the effects of the N127D mutationwithin M2.2 on biological activity, a series of point mutants weregenerated at this position. An alanine residue transforms wild-type NK1from an agonist into an antagonist by disrupting stabilizinginteractions of the NK1 homodimer. The effects of mutations to lysine orarginine at this position were tested. These substitutions introducesteric and electrostatic obstructions through bulky, chargedside-chains.

In addition, the point mutant D127N was analyzed; this reverts thisposition back to the wild-type asparagine residue. Within the context ofM2.2, which contains the N127D mutation, these mutations are referred toas D127A, D127K, D127R, and D127N. Importantly, each of these mutantsretained the high thermal stability associated with M2.2 (Table 5).

3.2 Characterization of NK1 mutants as Met receptor agonists orantagonists. The NK 1 mutants were evaluated in MDCK cell scatter anduPA asssays, two assays widely used to study activation of the Metreceptor in mammalian cells. For MDCK cell scatter assays, 1500cells/well were seeded into 96-well plates in 100 μL of complete growthmedia and incubated at 37° C., 5% CO₂. After 24 h, media was removed byaspiration and replaced with media containing HGF or NK1 proteins at aconcentration of 0.1 or 100 nM, respectively. In some experimentsLovenox® heparin (Sanofi-Aventis) was used at a concentration of 2 μM orat a 2:1 molar ratio of heparin:NK1. After 24 h, cells were fixed andstained with 0.5% crystal violet in 50% ethanol for 10 min at roomtemperature, washed with water, and dried in air prior to beingphotographed. MDCK scatter inhibition assays were performed is a similarmanner, except cells were incubated with 250 nM NK1 mutants for 30 minprior to adding HGF at a final concentration of 0.1 nM.

For MDCK uPA assays, 4000 cells/well were seeded into 96-well plates in100 μL of complete growth media and incubated at 37° C., 5% CO₂. After24 h, media was removed by aspiration and replaced with media containingHGF or NK1 at a concentration of 1 or 100 nM, respectively. After 24 h,cells were washed two times with 200 μL phenol red-free DMEM andincubated with 200 μL reaction buffer containing 50% (vol/vol) of 0.05units/mL plasminogen (Roche Applied Science), 40% (vol/vol) 50 mM TrispH 8.0, 10% (vol/vol) and 3 mM chromozym PL (Roche Applied Science) in100 mM glycine pH 3.5 solution. Plates were incubated for 4 h at 37° C.,5% CO₂ prior to measuring absorbance at 405 nm using an Infinite M1000microplate reader (Tecan Group Ltd.).

The mutants M2.2 D127A, D127K, and D127R did not induce Met activation,as measured by scatter (FIG. 10 and FIG. 11A) or uPA activation (FIG.11B) in MDCK cells. The unmodified M2.2 variant, which contains theN127D mulation, exhibited weak (FIG. 11A) or no agonistic activity (FIG.10 and FIG. 11B).

In contrast, reversion of position 127 to the wild-type asparagineresidue (M2.2 D127N) resulted in agonistic activity in both MDCK scatter(FIG. 10 and FIG. 11A) and uPA assays (FIG. 11B). The activity of M2.2D127N was similar to that of wild-type NK1, and both showed enhancedactivity in the presence of soluble heparing (FIG. 11C top vs. bottom).In comparison, M2.2D127A, D127K, and D127R did not exhibit agonisticactivity in these assays either in the presence of absence of heparin(FIG. 10 and FIG. 11A-C).

The ability of these mutants to inhibit HGF-induced Met activation wastested. As a control, M2.2 D127N did not inhibit HGF-induced activity,providing further evidence of its functions as a Met receptor agonist(FIG. 12). M2.2 mutants D127A, D127K, and D127R exhibited weak orminimal inhibition of HGF-induced MDCK scattering in the absence ofsoluble heparin (FIG. 12 top)

In contrast, strong antagonistic activity was observed with the additionof 2 μM heparin (FIG. 12 bottom). Pre-formulating the NK1 mutants with a2:1 molar ratio of heparin:NK1 was sufficient to confer thisantagonistic activity and obviated the need to add excess heparin forimproved antagonistic activity (FIG. 13). Unmodified M2.2 (M2.2 N127D)exhibited only weak antagonistic activity with a 2:1 molar ratio ofheparin (FIG. 13), supporting the utility of the rationally-engineeredpoint mutations. The antagonistic activity of M2.2 D127K is similar tothat of previously reported antagonist NK1 N127A (FIG. 13). However, theM2.2 D127A/K/and R mutants possess markedly improved stability andexpression compared to NK1 N127A, namely lower salt-dependent stability,an increased T_(m) of −15° C. and −40-fold improved recombinantexpression yield, which are all attractive properties.

4.1 Biochemical and biological characterization of recombinant Aras-4.Five of the clones from the third round of directed evolution wereselected for further investigation, based on their sequencedistribution, yeast surface expression level, and Met-Fc binding. Theseclones were referred to as Aras-1, -2, -3, -4, and -5 (FIG. 14). Each ofthese clones was found to be well expressed in the yeast Pichia pastorisexcept for Aras-1.

Aras-4 was selected for further characterization. It exhibited highthermal stability as determined by CD temperature melts (T_(m)=64.9±1.2°C.). Aras-4 does not activate cellular Met when added to a culture ofMDCK cells and effectively inhibited HGF-induced activation of Met atapproximately a five-fold lower concentration than M2.2 D127A or thewild-type NK1-based antagonist NK1 N127A (FIG. 15).

4.2 Introduction of Disulfide Linkages to Form Covalently Bound Dimers.A free cysteine residue was introduced to the N-terminus of M2.2 D127N,which resulted in the formation of monomeric and dimeric species uponrecombinant expression. The cystine-linked dimeric protein (termedcdD127N) was purified from the monomer using size-exclusionchromatography. SDS-PAGE analysis of cdD127N under reducing andnon-reducing conditions confirmed that a dimer is formed through acovalent disulfide bond. (FIG. 16). Cystine-linked dimeric M2.2 D127K(termed cdD127K) and Aras-4 (termed cdAras-4) polypeptides were alsogenerated.

4.3 Biological Activity of cdD127N, cdD127K, and cdAras-4. cdD127N andcdD127K exhibited agonistic activity at an order of magnitude lowerconcentration than the M2.2 D127N monomer which possesses similaragonistic activity to wild-type NK1 (FIG. 17). The agonist activity ofcdD127K is surprising since the parental monomer, M2.2 D127K, is anantagonist. Similarly surprising is the result for cdAras-4 wherein thecovalent linkage converted the antagonist Aras-4 into an agonist. Thelevel of agonistic activity observed is approaches that of full-lengthHGF, however cdD127N, cdD127K, and cdAras-4 possess substantiallyimproved stability relative to full length HGF and can be recombinantlyexpressed in yeast.

4.4 Only an N-terminal cysteine mediates homodimerization directly.Based on the crystal structure of NK1 homodimers, it was recognized thatposition 127 is in close proximity on adjacent protomers. This suggestedthe possibility of forming covalently linked homodimers by placing acysteine residue at this position. To test this possibility, a variantAras-4 polypeptide was generated in which the residue D127 wassubstituted with Cys. The resulting polypeptides largely failed toproduce dimers either spontaneously or after phenathroline-cupricsulfate treatment as shown in FIG. 18.

In addition to the covalent linkage through the addition of a freecysteine at the N-terminus of NK1 and variants, other locations andlinkers where tested. (FIG. 19). A free cysteine or a combination of afree cysteine with a cysteine tag (Backer et al. (2006) Nat. Med.13(4):504-509) were attached to the N-terminus or C-terminus of theAras-4 variant. Only the free cysteine at the N-terminus resulted indimeric protein upon recombinant expression in yeast.

5.0 Preparation of HGF Variant Polypeptides Containing Heparin AlginatePellets. Calcium alginate pellets may provide a stable platform for HGFbecause of enhanced retention of activity and storage time and thus canbe used as devices for controlled HGF variant release. Heparin-sepharosebeads (Pharmacia LKB) can be sterilized under ultraviolet light for 30minutes and then mixed with filter-sterilized sodium alginate. The mixedslurry can then be dropped through a needle into a beaker containing ahardened solution of CaCl₂ (1.5% wt/vol.). Beads can form instantly.Cross-linked capsule envelopes can be obtained by incubating thecapsules in the CaCl₂ solution for 5 minutes under gentle mixing andthen for 10 minutes without mixing. The formed beads can be washed withsterile water and stored in 0.9% NaCl-1 mmol/L CaCl₂ at 4° C. HGFloading may be performed by incubating 10 capsules in 0.9% NaCl-1 mmol/LCaCl₂-0.05% gelatin with 12.5 μg (for 10 μg dose) or 125 μg (for 100 μgdose) or HGF variant for 16 hours under gentle agitation at 4° C. Theend product may be sterilized under ultraviolet light for 30 minutes.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

All patents, patent applications, and other publications cited in thisapplication are incorporated by reference in their entirety.

1.-13. (canceled)
 14. A dimer comprising a first human NK1 (hNK1)variant polypeptide and a second hNK1 variant polypeptide, wherein thefirst hNK1 variant polypeptide comprises a first linker and the secondhNK1 polypeptide comprises a second linker, wherein the first linkercomprises at least one cysteine residue and the second linker comprisesa moiety reactive with a sulfhydryl group of the cysteine, wherein thedimer is formed by the reaction of cysteine with the moiety reactivewith the sulfhydryl group. 15.-17. (canceled)
 18. A method of tissueregeneration, said method comprising contacting cells with an effectiveamount of the dimer according to claim
 14. 19. A pharmaceuticalformulation comprising the dimer of claim 14 and a pharmaceuticallyacceptable carrier.
 20. The dimer according to claim 14, which is anagonist of Met.
 21. The dimer according to claim 14, which is ahomodimer.
 22. The dimer according to claim 14, wherein at least one ofsaid first and second NK1 variant polypeptides includes an amino acidsubstitution at amino acid position 62, 95, 132, 137, 170, 173, or 193.23. The dimer according to claim 14, wherein at least one of said firstand second NK1 variant polypeptides includes an amino acid substitutionselected from K137R, K170E, N193D, K62E, Q173R, Q95R, K132N/R, andcombinations thereof.
 24. The dimer according to claim 14, wherein atleast one of said first and second NK1 variant polypeptides comprisesthe amino acid substitution K62E.
 25. The dimer according to claim 14,wherein at least one of said first and second NK1 variant polypeptidescomprises the amino acid substitution Q95R.
 26. The dimer according toclaim 14, wherein at least one of said first and second NK1 variantpolypeptides comprises the amino acid substitution K132N.
 27. The dimeraccording to claim 14, wherein at least one of said first and second NK1variant polypeptides comprises the amino acid substitution K137R. 28.The dimer according to claim 14, wherein at least one of said first andsecond NK1 variant polypeptides comprises the amino acid substitutionK170E.
 29. The dimer according to claim 14, wherein at least one of saidfirst and second NK1 variant polypeptides comprises the amino acidsubstitution Q173R.
 30. The dimer according to claim 14, wherein atleast one of said first and second NK1 variant polypeptides comprisesthe amino acid substitution N193D.
 31. The dimer according to claim 14,wherein at least one of said first and second NK1 variant polypeptidescomprises NK1 variant M2.2 D127N.
 32. The dimer according to claim 14,wherein both of said first and second NK1 variant polypeptides comprisesNK1 variant M2.2 D127N.
 33. The dimer according to claim 14, wherein atleast one of said first and second NK1 variant polypeptides comprisesNK1 variant M2.2 D127K.
 34. The dimer according to claim 14, whereinboth of said first and second NK1 variants of said dimer comprises NK1variant M2.2 D127K.
 35. The dimer according to claim 14, which iscdD127N.
 36. The dimer according to claim 14, which is cdD127K.